Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo

Assessment of an in vitro whole cigarette smoke exposure system: The Borgwaldt RM20S 8-syringe smoking machine

<p>Abstract</p> <p>Background</p> <p>There have been many recent developments of <it>in vitro </it>cigarette smoke systems closely replicating <it>in vivo </it>exposures. The Borgwaldt RM20S smoking machine (RM20S) enables the serial dilution and delivery of cigarette smoke to exposure chambers for <it>in vitro </it>analyses. In this study we have demonstrated reliability and robustness testing of the RM20S in delivering smoke to <it>in vitro </it>cultures using an in-house designed whole smoke exposure chamber.</p> <p>Results</p> <p>The syringe precision and accuracy of smoke dose generated by the RM20S was assessed using a methane gas standard and resulted in a repeatability error of ≤9%. Differential electrical mobility particle spectrometry (DMS) measured smoke particles generated from reference 3R4F cigarettes at points along the RM20S. 53% ± 5.9% of particles by mass reached the chamber, the remainder deposited in the syringe or connecting tubing and ~16% deposited in the chamber. Spectrofluorometric quantification of particle deposition within chambers indicated a positive correlation between smoke concentration and particle deposition. <it>In vitro </it>air-liquid interface (ALI) cultures (H292 lung epithelial cells), exposed to whole smoke (1:60 dilution (smoke:air, equivalent to ~5 μg/cm²)) demonstrated uniform smoke delivery within the chamber.</p> <p>Conclusions</p> <p>These results suggest this smoke exposure system is a reliable and repeatable method of generating and exposing ALI <it>in vitro </it>cultures to cigarette smoke. This system will enable the evaluation of future tobacco products and individual components of cigarette smoke and may be used as an alternative <it>in vitro </it>tool for evaluating other aerosols and gaseous mixtures such as air pollutants, inhaled pharmaceuticals and cosmetics.</p

Expression signatures of TP53 mutations in serous ovarian cancers

<p>Abstract</p> <p>Background</p> <p>Mutations in the <it>TP53 </it>gene are extremely common and occur very early in the progression of serous ovarian cancers. Gene expression patterns that relate to mutational status may provide insight into the etiology and biology of the disease.</p> <p>Methods</p> <p>The <it>TP53 </it>coding region was sequenced in 89 frozen serous ovarian cancers, 40 early stage (I/II) and 49 advanced stage (III/IV). Affymetrix U133A expression data was used to define gene expression patterns by mutation, type of mutation, and cancer stage.</p> <p>Results</p> <p>Missense or chain terminating (null) mutations in <it>TP53 </it>were found in 59/89 (66%) ovarian cancers. Early stage cancers had a significantly higher rate of null mutations than late stage disease (38% vs. 8%, p < 0.03). In advanced stage cases, mutations were more prevalent in short term survivors than long term survivors (81% vs. 30%, p = 0.0004). Gene expression patterns had a robust ability to predict <it>TP53 </it>status within training data. By using early versus late stage disease for out of sample predictions, the signature derived from early stage cancers could accurately (86%) predict mutation status of late stage cancers.</p> <p>Conclusions</p> <p>This represents the first attempt to define a genomic signature of <it>TP53 </it>mutation in ovarian cancer. Patterns of gene expression characteristic of <it>TP53 </it>mutation could be discerned and included several genes that are known p53 targets or have been described in the context of expression signatures of <it>TP53 </it>mutation in breast cancer.</p

N-Octanoyl-Dopamine inhibits cytokine production in activated T-cells and diminishes MHC-class-II expression as well as adhesion molecules in IFN gamma-stimulated endothelial cells

IFN gamma enhances allograft immunogenicity and facilitates T-cell mediated rejection. This may cause interstitial fibrosis and tubular atrophy (IFTA), contributing to chronic allograft loss. We assessed if inhibition of T-cell activation by N-octanoyl dopamine (NOD) impairs adherence of activated T-cells to endothelial cells and the ability of activated T-cells to produce IFN gamma. We also assessed if NOD affects IFN gamma mediated gene expression in endothelial cells. The presence of NOD during T-cell activation significantly blunted their adhesion to unstimulated and cytokine stimulated HUVEC. Supernatants of these T-cells displayed significantly lower concentrations of TNF alpha and IFN gamma and were less capable to facilitate T-cell adhesion. In the presence of NOD VLA-4 (CD49d/CD29) and LFA-1 (CD11a/CD18) expression on T-cells was reduced. NOD treatment of IFN gamma stimulated HUVEC reduced the expression of MHC class II transactivator (CIITA), of MHC class II and its associated invariant chain CD74. Since IFTA is associated with T-cell mediated rejection and IFN gamma to a large extent regulates immunogenicity of allografts, our current data suggest a potential clinical use of NOD in the treatment of transplant recipients. Further in vivo studies are warranted to confirm these in vitro findings and to assess the benefit of NOD on IFTA in clinically relevant models

Mutant p53 drives multinucleation and invasion through a process that is suppressed by ANKRD11

Mutations of p53 in cancer can result in a gain of function associated with tumour progression and metastasis. We show that inducible expression of several p53 ‘hotspot’ mutants promote a range of centrosome abnormalities, including centrosome amplification, increased centrosome size and loss of cohesion, which lead to mitotic defects and multinucleation. These mutant p53-expressing cells also show a change in morphology and enhanced invasive capabilities. Consequently, we sought for a means to specifically target the function of mutant p53 in cancer cells. This study has identified ANKRD11 as a key regulator of the oncogenic potential of mutant p53. Loss of ANKRD11 expression with p53 mutation defines breast cancer patients with poor prognosis. ANKRD11 alleviates the mitotic defects driven by mutant p53 and suppresses mutant p53-mediated mesenchymal-like transformation and invasion. Mechanistically, we show that ANKRD11 restores a native conformation to the mutant p53 protein and causes dissociation of the mutant p53–p63 complex. This represents the first evidence of an endogenous protein with the capacity to suppress the oncogenic properties of mutant p53.JE Noll, J Jeffery, F Al-Ejeh, R Kumar, KK Khanna, DF Callen and PM Neilse

Mutant p53 gain-of-function induces epithelial–mesenchymal transition through modulation of the miR-130b–ZEB1 axis

The tumor suppressor gene p53 has been implicated in the regulation of epithelial–mesenchymal transition (EMT) and tumor metastasis by regulating microRNA (miRNA) expression. Here, we report that mutant p53 exerts oncogenic functions and promotes EMT in endometrial cancer (EC) by directly binding to the promoter of miR-130b (a negative regulator of ZEB1) and inhibiting its transcription. We transduced p53 mutants into p53-null EC cells, profiled the miRNA expression by miRNA microarray and identified miR-130b as a potential target of mutant p53. Ectopic expression of p53 mutants repressed the expression of miR-130b and triggered ZEB1-dependent EMT and cancer cell invasion. Loss of an endogenous p53 mutation increased the expression of miR-130b, which resulted in reduced ZEB1 expression and attenuation of the EMT phenotype. Furthermore, re-expression of miR-130b suppressed mutant p53-induced EMT and ZEB1 expression. Importantly, the expression of miR-130 was significantly reduced in EC tissues, and patients with higher expression levels of miR-130b survived longer. These data provide a novel understanding of the roles of p53 gain-of-function mutations in accelerating tumor progression and metastasis through modulation of the miR-130b–ZEB1 axis