Inhibiting the phosphatidylinositide 3-kinase pathway blocks radiation-induced metastasis associated with Rho-GTPase and Hypoxia-inducible factor-1 activity

AbstractBackground and purposeUndifferentiated follicular and anaplastic thyroid tumours often respond poorly to radiotherapy and show increased metastatic potential. We evaluated radiation-induced effects on metastasis in thyroid carcinoma cells and tumours, mechanistically focusing on phosphatidylinositide 3-kinase (PI3K) and associated pathways.Material and methodsMigration was analysed in follicular (FTC133) and anaplastic (8505c) cells following radiotherapy (0–6 Gray) with concomitant pharmacological (GDC-0941) or genetic inhibition of PI3K. Hypoxia-inducible factor-1 (HIF-1)-activity was measured using luciferase reporter assays and was inhibited using a dominant-negative variant. Activation and subcellular localisation of target proteins were assessed via Western blot and immunofluorescence. In vivo studies used FTC133 xenografts with metastatic lung dissemination assessed ex vivo.ResultsRadiation induced migration in a HIF-dependent manner in FTC133 cells but decreased migration in 8505c’s. Post-radiation HIF-activity correlated with migratory phenotype. PI3K-targeting inhibited migration under basal and irradiated conditions through inhibition of HIF-1α, Rho-GTPase expression/activity and localisation whilst having little effect on src/FAK. In vivo, radiation induced PI3K, HIF, Rho-GTPases and src but only PI3K, HIF and Rho-GTPases were inhibited by GDC-0941. Co-treatment with GDC-0941 and radiation significantly reduced metastatic dissemination versus radiotherapy alone.ConclusionsRadiation modifies metastatic characteristics of thyroid carcinoma cells, which can be successfully inhibited by targeting PI3K using GDC-0941 in vitro and in vivo

Hypoxia-inducible factor in thyroid carcinoma.

Intratumoural hypoxia (low oxygen tension) is associated with aggressive disease and poor prognosis. Hypoxia-inducible factor-1 is a transcription factor activated by hypoxia that regulates the expression of genes that promote tumour cell survival, progression, metastasis, and resistance to chemo/radiotherapy. In addition to hypoxia, HIF-1 can be activated by growth factor-signalling pathways such as the mitogen-activated protein kinases- (MAPK-) and phosphatidylinositol-3-OH kinases- (PI3K-) signalling cascades. Mutations in these pathways are common in thyroid carcinoma and lead to enhanced HIF-1 expression and activity. Here, we summarise current data that highlights the potential role of both hypoxia and MAPK/PI3K-induced HIF-1 signalling in thyroid carcinoma progression, metastatic characteristics, and the potential role of HIF-1 in thyroid carcinoma response to radiotherapy. Direct or indirect targeting of HIF-1 using an MAPK or PI3K inhibitor in combination with radiotherapy may be a new potential therapeutic target to improve the therapeutic response of thyroid carcinoma to radiotherapy and reduce metastatic burden

The immunogenicity of a viral cytotoxic T cell epitope is controlled by its MHC-bound conformation

Thousands of potentially antigenic peptides are encoded by an infecting pathogen; however, only a small proportion induce measurable CD8+ T cell responses. To investigate the factors that control peptide immunogenicity, we have examined the cytotoxic T lymphocyte (CTL) response to a previously undefined epitope (77APQPAPENAY86) from the BZLF1 protein of Epstein-Barr virus (EBV). This peptide binds well to two human histocompatibility leukocyte antigen (HLA) allotypes, HLA-B*3501 and HLA-B*3508, which differ by a single amino acid at position 156 (156Leucine vs. 156Arginine, respectively). Surprisingly, only individuals expressing HLA-B*3508 show evidence of a CTL response to the 77APQPAPENAY86 epitope even though EBV-infected cells expressing HLA-B*3501 process and present similar amounts of peptide for CTL recognition, suggesting that factors other than peptide presentation levels are influencing immunogenicity. Functional and structural analysis revealed marked conformational differences in the peptide, when bound to each HLA-B35 allotype, that are dictated by the polymorphic HLA residue 156 and that directly affected T cell receptor recognition. These data indicate that the immunogenicity of an antigenic peptide is influenced not only by how well the peptide binds to major histocompatibility complex (MHC) molecules but also by its bound conformation. It also illustrates a novel mechanism through which MHC polymorphism can further diversify the immune response to infecting pathogens

Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20).

The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours.Thanks to Dr John Bomalaski, (Polaris Pharmaceuticals, Inc) for supplying the ADI-PEG20, to Dr Simon S Hoer for useful discussions and to members of Histopathology/ISH (CRUK Cambridge Institute, UK) for IHC and imaging assistance. This work was supported by the Wellcome Trust and the NIHR Cambridge Biomedical Research Centre Senior Investigator Awards (to P.H.M., supporting N.B.), EU FP7 Metoxia Grant agreement no. 222741 (to P.H.M., supporting G.C.), UCL Cancer Research UK Centre (to M.R.), King’s College London and UCL Comprehensive Cancer Imaging Centre, Cancer Research UK and EPSRC in association with the Medical Research Council (MRC), the DoH (England: to R.B.P.), MRC Cancer Unit Core Funding (to C.F., supporting E.G.).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep2295

Discovery of an Ultrasoft X-ray Transient Source in the 2XMM Catalog: a Tidal Disruption Event Candidate

We have discovered an ultrasoft X-ray transient source, 2XMMi J184725.1-631724, which was detected serendipitously in two XMM-Newton observations in the direction of the center of the galaxy IC 4765-f01-1504 at a redshift of 0.0353. These two observations were separated by 211 days, with the 0.2-10 keV absorbed flux increasing by a factor of about 9. Their spectra are best described by a model dominated by a thermal disk or a single-temperature blackbody component (contributing >80% of the flux) plus a weak power-law component. The thermal emission has a temperature of a few tens of eV, and the weak power-law component has a photon index of ~3.5. Similar to the black hole X-ray binaries in the thermal state, our source exhibits an accretion disk whose luminosity appears to follow the $L\propto T^4$ relation. This would indicate that the black hole mass is about 10^5-10^6 M_sun using the best-fitting inner disk radius. Both XMM-Newton observations show variability of about 21% on timescales of hours, which can be explained as due to fast variations in the mass accretion rate. The source was not detected by ROSAT in an observation in 1992, indicating a variability factor of >64 over longer timescales. The source was not detected again in X-rays in a Swift observation in 2011 February, implying a flux decrease by a factor of >12 since the last XMM-Newton observation. The transient nature, in addition to the extreme softness of the X-ray spectra and the inactivity of the galaxy implied by the lack of strong optical emission lines, makes it a candidate tidal disruption event. If this is the case, the first XMM-Newton observation would have been in the rising phase, and the second one in the decay phase.Comment: 12 pages, 6 figures. Accepted for publication in Ap

Carbon on the Northwest European Shelf: Contemporary Budget and Future Influences

A carbon budget for the northwest European continental shelf seas (NWES) was synthesized using available estimates for coastal, pelagic and benthic carbon stocks and flows. Key uncertainties were identified and the effect of future impacts on the carbon budget were assessed. The water of the shelf seas contains between 210 and 230 Tmol of carbon and absorbs between 1.3 and 3.3 Tmol from the atmosphere annually. Off-shelf transport and burial in the sediments account for 60–100 and 0–40% of carbon outputs from the NWES, respectively. Both of these fluxes remain poorly constrained by observations and resolving their magnitudes and relative importance is a key research priority. Pelagic and benthic carbon stocks are dominated by inorganic carbon. Shelf sediments contain the largest stock of carbon, with between 520 and 1600 Tmol stored in the top 0.1 m of the sea bed. Coastal habitats such as salt marshes and mud flats contain large amounts of carbon per unit area but their total carbon stocks are small compared to pelagic and benthic stocks due to their smaller spatial extent. The large pelagic stock of carbon will continue to increase due to the rising concentration of atmospheric CO2, with associated pH decrease. Pelagic carbon stocks and flows are also likely to be significantly affected by increasing acidity and temperature, and circulation changes but the net impact is uncertain. Benthic carbon stocks will be affected by increasing temperature and acidity, and decreasing oxygen concentrations, although the net impact of these interrelated changes on carbon stocks is uncertain and a major knowledge gap. The impact of bottom trawling on benthic carbon stocks is unique amongst the impacts we consider in that it is widespread and also directly manageable, although its net effect on the carbon budget is uncertain. Coastal habitats are vulnerable to sea level rise and are strongly impacted by management decisions. Local, national and regional actions have the potential to protect or enhance carbon storage, but ultimately global governance, via controls on emissions, has the greatest potential to influence the long-term fate of carbon stocks in the northwestern European continental shelf

Phosphatidylinositide 3-kinase (PI3K) and PI3K-related kinase (PIKK) activity contributes to radioresistance in thyroid carcinomas.

Anaplastic (ATC) and certain follicular thyroid-carcinomas (FTCs) are radioresistant. The Phosphatidylinositide 3-kinase (PI3K) pathway is commonly hyperactivated in thyroid-carcinomas. PI3K can modify the PI3K-related kinases (PIKKs) in response to radiation: How PIKKs interact with PI3K and contribute to radioresistance in thyroid-carcinomas is unknown. Further uncertainties exist in how these interactions function under the radioresistant hypoxic microenvironment. Under normoxia/anoxia, ATC (8505c) and FTC (FTC-133) cells were irradiated, with PI3K-inhibition (via GDC-0941 and PTEN-reconstitution into PTEN-null FTC-133s) and effects on PIKK-activation, DNA-damage, clonogenic-survival and cell cycle, assessed. FTC-xenografts were treated with 5 × 2 Gy, ± 50 mg/kg GDC-0941 (twice-daily; orally) for 14 days and PIKK-activation and tumour-growth assessed. PIKK-expression was additionally assessed in 12 human papillary thyroid-carcinomas, 13 FTCs and 12 ATCs. GDC-0941 inhibited radiation-induced activation of Ataxia-telangiectasia mutated (ATM), ATM-and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inhibition of ATM and DNA-PKcs was PI3K-dependent, since activation was reduced in PTEN-reconstituted FTC-133s. Inhibition of PIKK-activation was greater under anoxia: Consequently, whilst DNA-damage was increased and prolonged under both normoxia and anoxia, PI3K-inhibition only reduced clonogenic-survival under anoxia. GDC-0941 abrogated radiation-induced cell cycle arrest, an effect most likely linked to the marked inhibition of ATR-activation. Importantly, GDC-0941 inhibited radiation-induced PIKK-activation in FTC-xenografts leading to a significant increase in time taken for tumours to triple in size: 26.5 ± 5 days (radiation-alone) versus 31.5 ± 5 days (dual-treatment). PIKKs were highly expressed across human thyroid-carcinoma classifications, with ATM scoring consistently lower. Interestingly, some loss of ATM and DNA-PKcs was observed. These data provide new insight into the mechanisms of hypoxia-associated radioresistance in thyroid-carcinoma

The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein.

BACKGROUND: Regions within solid tumours often experience oxygen deprivation, which is associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitising effect of gemcitabine and its main metabolite dFdU under normoxia versus hypoxia and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitising mechanism. METHODS: Stable expression of dominant negative HIF-1α (dnHIF) in MDA-MB-231 breast cancer cells, that ablated endogenous HIF-1 transcriptional activity, was validated by western blot and functionality was assessed by HIF-1α activity assay. Cells were exposed to varying oxygen environments and treated with gemcitabine or dFdU for 24 h, followed by irradiation. Clonogenicity was then assessed. Using radiosensitising conditions, cells were collected for cell cycle analysis. RESULTS: HIF-1 activity was significantly inhibited in cells stably expressing dnHIF. A clear radiosensitising effect under normoxia and hypoxia was observed for both gemcitabine and dFdU. No significant difference in radiobiological parameters between HIF-1 proficient and HIF-1 deficient MDA-MB-231 cells was demonstrated. CONCLUSIONS: For the first time, radiosensitisation by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. No major role for functional HIF-1 protein in radiosensitisation by gemcitabine or dFdU could be shown

The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein.

BACKGROUND: Regions within solid tumours often experience oxygen deprivation, which is associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitising effect of gemcitabine and its main metabolite dFdU under normoxia versus hypoxia and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitising mechanism. METHODS: Stable expression of dominant negative HIF-1α (dnHIF) in MDA-MB-231 breast cancer cells, that ablated endogenous HIF-1 transcriptional activity, was validated by western blot and functionality was assessed by HIF-1α activity assay. Cells were exposed to varying oxygen environments and treated with gemcitabine or dFdU for 24 h, followed by irradiation. Clonogenicity was then assessed. Using radiosensitising conditions, cells were collected for cell cycle analysis. RESULTS: HIF-1 activity was significantly inhibited in cells stably expressing dnHIF. A clear radiosensitising effect under normoxia and hypoxia was observed for both gemcitabine and dFdU. No significant difference in radiobiological parameters between HIF-1 proficient and HIF-1 deficient MDA-MB-231 cells was demonstrated. CONCLUSIONS: For the first time, radiosensitisation by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. No major role for functional HIF-1 protein in radiosensitisation by gemcitabine or dFdU could be shown

Supplemental data 1 Shape analysis

The diameters of our nanoparticles are determined by using the free online image analysis tool ImageJ [1] which includes a particle analysis package. We use the 2D images taken in STEM mode to measure the surface area A of the nanoparticle, whereafter we determine the mean nanoparticle diameter D using the relation A = π ( D /2) 2 . The particle analysis tool also evaluates the maximum D max and minimum D min diameters of the nanoparticle and the difference between these two diameters, i.e., Δ D = D max -D min provides us a measure for error in the nanoparticle diameter (shown as the error bar in Article Supplementary In order to understand the scattering of the SP resonance energies observed in Articl