Integrative characterisation of secreted factors involved in intercellular communication between prostate epithelial or cancer cells and fibroblasts

Reciprocal interactions between prostate cancer cells and carcinomaassociated fibroblasts (CAFs) mediate cancer development and progression; however, our understanding of the signalling pathways mediating these cellular interactions remains incomplete. To address this, we defined secretome changes upon co-culture of prostate epithelial or cancer cells with fibroblasts that mimic bi-directional communication in tumours. Using antibody arrays, we profiled conditioned media from mono- and cocultures of prostate fibroblasts, epithelial and cancer cells, identifying secreted proteins that are upregulated in co-culture compared to monoculture. Six of these (CXCL10, CXCL16, CXCL6, FST, PDGFAA, IL17B) were functionally screened by siRNA knockdown in prostate cancer cell/fibroblast co-cultures, revealing a key role for follistatin (FST), a secreted glycoprotein that binds and bioneutralises specific members of the TGF-b superfamily, including activin A. Expression of FST by both cell types was required for the fibroblasts to enhance prostate cancer cell proliferation and migration, whereas FST knockdown in co-culture grafts decreased tumour growth in mouse xenografts. This study highlights the complexity of prostate cancer cell–fibroblast communication, demonstrates that co-culture secretomes cannot be predicted from individual cultures, and identifies FST as a tumour-microenvironment-derived secreted factor that represents a candidate therapeutic target.Yunjian Wu, Kimberley C. Clark, Birunthi Niranjan, Anderly C. Chueh, Lisa G. Horvath, Renea A. Taylor, and Roger J. Dal

Proteomic characterisation of prostate cancer intercellular communication reveals cell type-selective signalling and TMSB4X-dependent fibroblast reprogramming

Background: In prostate cancer, the tumour microenvironment (TME) represents an important regulator of disease progression and response to treatment. In the TME, cancer-associated fbroblasts (CAFs) play a key role in tumour progression, however the mechanisms underpinning fbroblast-cancer cell interactions are incompletely resolved. Here, we address this by applying cell type-specifc labelling with amino acid precursors (CTAP) and mass spectrometry (MS)-based (phospho) proteomics to prostate cancer for the frst time. Methods: Reciprocal interactions between PC3 prostate cancer cells co-cultured with WPMY-1 prostatic fbroblasts were characterised using CTAP-MS. Signalling network changes were determined using Metascape and Enrichr and visualised using Cytoscape. Thymosin β4 (TMSB4X) overexpression was achieved via retroviral transduction and assayed by ELISA. Cell motility was determined using Transwell and random cell migration assays and expression of CAF markers by indirect immunofuorescence. Results: WPMY-1 cells co-cultured with PC3s demonstrated a CAF-like phenotype, characterised by enhanced PDGFRB expression and alterations in signalling pathways regulating epithelial-mesenchymal transition, cytoskeletal organisation and cell polarisation. In contrast, co-cultured PC3 cells exhibited more modest network changes, with alterations in mTORC1 signalling and regulation of the actin cytoskeleton. The expression of the actin binding protein TMSB4X was signifcantly decreased in co-cultured WPMY-1 fbroblasts, and overexpression of TMSB4X in fbroblasts decreased migration of cocultured PC3 cells, reduced fbroblast motility, and protected the fbroblasts from being educated to a CAF-like phenotype by prostate cancer cells. Conclusions: This study highlights the potential of CTAP-MS to characterise intercellular communication within the prostate TME and identify regulators of cellular crosstalk such as TMSB4X.Yunjian Wu, Kimberley C. Clark, Elizabeth V. Nguyen, Birunthi Niranjan, Lisa G. Horvath, Renea A. Taylor, Roger J. Dal

The influence of ortho- and para-diphenoloxidase substrates on pigment formation in black yeast-like fungi

Dothideaceous black yeast-like fungi (BYF) are known to synthesise DHN-melanin that is inhibited by the systemic fungicide tricyclazole. The final step of the DHN melanin pathway is the conjoining of 1,8-DHN molecules to form the melanin polymer. There are several candidate enzymes for this step, including phenoloxidases such as tyrosinase and laccases, peroxidases, and perhaps also catalases. We analysed the type polyphenoloxidases that are involved in biosynthesis of BYF melanins. For that purpose we used substrates of o-diphenoloxidases (EC 1.10.3.1.): 4-hydroxyphenyl-pyruvic acid, L-β-phenyllactic acid, tyrosine, pyrocatechol, 3,4-dihydroxyphenylalanine and homogentisic acid, as well as substrates of p-diphenoloxidases (EC 1.10.3.2.): syringaldazine, resorcinol, p-phenylenediamine, phloroglucinol, guaiacol and pyrogallic acid. Fourteen strains of black yeasts originating from different natural biotopes were investigated. The tested strains could be divided into four groups based on their ability to produce dark pigments when cultivated on aromatic substrates of o- and on p-diphenoloxidases. It was established that syringaldazine, pyrogallic acid and 4-hydrophenyl-pyruvic acid, β-phenyllactic acid optimally promote melanin biosynthesis. Average intensity of pigmentation of all strains studied was minimal when guaiacol was used as a substrate. The present investigation indicates that the melanisation process may involve more enzymes and more substrates than those commonly recognised. Black yeasts are likely to contain a multipotent polyphenoloxidase

Measurement of the polarisation of W bosons produced with large transverse momentum in pp collisions at sqrt(s) = 7 TeV with the ATLAS experiment

This paper describes an analysis of the angular distribution of W->enu and W->munu decays, using data from pp collisions at sqrt(s) = 7 TeV recorded with the ATLAS detector at the LHC in 2010, corresponding to an integrated luminosity of about 35 pb^-1. Using the decay lepton transverse momentum and the missing transverse energy, the W decay angular distribution projected onto the transverse plane is obtained and analysed in terms of helicity fractions f0, fL and fR over two ranges of W transverse momentum (ptw): 35 < ptw < 50 GeV and ptw > 50 GeV. Good agreement is found with theoretical predictions. For ptw > 50 GeV, the values of f0 and fL-fR, averaged over charge and lepton flavour, are measured to be : f0 = 0.127 +/- 0.030 +/- 0.108 and fL-fR = 0.252 +/- 0.017 +/- 0.030, where the first uncertainties are statistical, and the second include all systematic effects.Comment: 19 pages plus author list (34 pages total), 9 figures, 11 tables, revised author list, matches European Journal of Physics C versio

Observation of a new chi_b state in radiative transitions to Upsilon(1S) and Upsilon(2S) at ATLAS

The chi_b(nP) quarkonium states are produced in proton-proton collisions at the Large Hadron Collider (LHC) at sqrt(s) = 7 TeV and recorded by the ATLAS detector. Using a data sample corresponding to an integrated luminosity of 4.4 fb^-1, these states are reconstructed through their radiative decays to Upsilon(1S,2S) with Upsilon->mu+mu-. In addition to the mass peaks corresponding to the decay modes chi_b(1P,2P)->Upsilon(1S)gamma, a new structure centered at a mass of 10.530+/-0.005 (stat.)+/-0.009 (syst.) GeV is also observed, in both the Upsilon(1S)gamma and Upsilon(2S)gamma decay modes. This is interpreted as the chi_b(3P) system.Comment: 5 pages plus author list (18 pages total), 2 figures, 1 table, corrected author list, matches final version in Physical Review Letter

Search for displaced vertices arising from decays of new heavy particles in 7 TeV pp collisions at ATLAS

We present the results of a search for new, heavy particles that decay at a significant distance from their production point into a final state containing charged hadrons in association with a high-momentum muon. The search is conducted in a pp-collision data sample with a center-of-mass energy of 7 TeV and an integrated luminosity of 33 pb^-1 collected in 2010 by the ATLAS detector operating at the Large Hadron Collider. Production of such particles is expected in various scenarios of physics beyond the standard model. We observe no signal and place limits on the production cross-section of supersymmetric particles in an R-parity-violating scenario as a function of the neutralino lifetime. Limits are presented for different squark and neutralino masses, enabling extension of the limits to a variety of other models.Comment: 8 pages plus author list (20 pages total), 8 figures, 1 table, final version to appear in Physics Letters

Measurement of the inclusive isolated prompt photon cross-section in pp collisions at sqrt(s)= 7 TeV using 35 pb-1 of ATLAS data

A measurement of the differential cross-section for the inclusive production of isolated prompt photons in pp collisions at a center-of-mass energy sqrt(s) = 7 TeV is presented. The measurement covers the pseudorapidity ranges |eta|<1.37 and 1.52<=|eta|<2.37 in the transverse energy range 45<=E_T<400GeV. The results are based on an integrated luminosity of 35 pb-1, collected with the ATLAS detector at the LHC. The yields of the signal photons are measured using a data-driven technique, based on the observed distribution of the hadronic energy in a narrow cone around the photon candidate and the photon selection criteria. The results are compared with next-to-leading order perturbative QCD calculations and found to be in good agreement over four orders of magnitude in cross-section.Comment: 7 pages plus author list (18 pages total), 2 figures, 4 tables, final version published in Physics Letters