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Ashida S.,Cleveland Clinic | Orloff M.S.,Cleveland Clinic | Orloff M.S.,Taussig Cancer Institute | Bebek G.,Cleveland Clinic | And 10 more authors.
Clinical Cancer Research

Purpose: Recent studies suggest that tumor microenvironment (stroma) is important in carcinogenesis and progression. We sought to integrate global genomic structural and expressional alterations in prostate cancer epithelium and stroma and their association with clinicopathologic features. Experimental Design: We conducted a genome-wide LOH/allelic imbalance (AI) scan of DNA from epithelium and stroma of 116 prostate cancers. LOH/AI hot or cold spots were defined as the markers with significantly higher or lower LOH/AI frequencies compared with the average frequency for markers along the same chromosome. These data were then integrated with publicly available transcriptome data sets and our experimentally derived data. Immunohistochemistry on an independent series was used for validation. Results: Overall, we identified 43 LOH/AI hot/cold spots, 17 in epithelium and stroma (P < 0.001), 18 only in epithelium (P < 0.001), and eight only in stroma (P < 0.001). Hierarchical clustering of expression data supervised by genes within LOH/AI hot/cold spots in both epithelium and stroma accurately separated samples into normal epithelium, primary cancer, and metastatic cancer groups, which could not be achieved with data from only epithelium. Importantly, our experimental expression data of the genes within the LOH/AI hot/cold spots in stroma accurately clustered normal stroma from cancer stroma. We also identified 15 LOH/AI markers that were associated with Gleason score, which were validated functionally in each compartment by transcriptome data. Independent immunohistochemical validation of STIM2 within a stromal significantLOH marker (identified as associated with Gleason grade) confirmed its downregulation in the transition from moderate to high Gleason grade. Conclusions: Compartment-specific genomic and transcriptomic alterations accurately distinguish clinical and pathologic outcomes, suggesting new biomarkers for prognosis and targeted therapeutics. © 2012 AACR. Source

Salanga C.L.,University of California at San Diego | Dyer D.P.,University of California at San Diego | Kiselar J.G.,Center for Proteomics and Bioinformatics | Gupta S.,Center for Proteomics and Bioinformatics | And 4 more authors.
Journal of Biological Chemistry

The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP- 3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAGbinding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate thatCCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Source

Schlatzer D.M.,Center for Proteomics and Bioinformatics | Sugalski J.M.,Case Western Reserve University | Chen Y.,Case Western Reserve University | Barnholtz-Sloan J.,Case Western Reserve University | And 9 more authors.
Journal of Acquired Immune Deficiency Syndromes

Background: HIV infection contributes to accelerated rates of progression of liver fibrosis during hepatitis C virus (HCV) infection, and HCV liver disease contributes to mortality during HIV infection. Although mechanisms underlying these interactions are not well known, soluble and cellular markers of immune activation associate with disease progression during both infections. Methods: We identified proteins varying in expression across the plasma proteomes of subjects with untreated HIV infection, untreated HCV infection with low aspartate transaminase/platelet ratio index, untreated HCV infection with high aspartate transaminase/ platelet ratio index, HIV-HCV coinfection, and controls. We examined correlations between dysregulated proteins and markers of immune activation to uncover biomarkers specific to disease states. Results: We observed the anticipated higher frequencies of HLADR+ CD38 +CD4 and CD8 T cells, higher serum soluble CD14 levels, and higher serum interleukin-6 levels for HCV- and HIV-infected groups compared with controls. Plasma proteome analysis identified 2297 peptides mapping to 227 proteins, and quantitative analysis of peptide intensity identified significant changes in 85 proteins across the 5 groups. Abundance for 7 of these proteins was validated by enzyme-linked immunosorbent assay. Forty-three of these proteins correlated with markers of immune activation, including at least 2 proteins that may directly drive T-cell activation. As a functional validation, we tested the enzymatic pathway product (lysophosphatidic acid, LPA) of one such protein, ecotonucleotide pyrophosphatase/ phosphodiesterase-2, for ability to activate T cells in vitro. LPA activated T cells to express CD38 and HLA-DR. Conclusions: These data indicate that elevated levels of ecotonucleotide pyrophosphatase/phosphodiesterase-2 and LPA during advanced HCV disease may play a role in exacerbating immune activation during HCV-HIV coinfection. Copyright © 2013 by Lippincott Williams & Wilkins. Source

Tomechko S.E.,Center for Proteomics and Bioinformatics | Liu G.,Case Western Reserve University | Tao M.,Case Western Reserve University | Schlatzer D.,Center for Proteomics and Bioinformatics | And 4 more authors.
Molecular and Cellular Proteomics

Diabetes mellitus is well known to cause bladder dysfunction; however, the molecular mechanisms governing this process and the effects on individual tissue elements within the bladder are poorly understood, particularly in type 2 diabetes. A shotgun proteomics approach was applied to identify proteins differentially expressed between type 2 diabetic (TallyHo) and control (SWR/J) mice in the bladder smooth muscle and urothelium, separately. We were able to identify 1760 nonredundant proteins from the detrusor smooth muscle and 3169 nonredundant proteins from urothelium. Pathway and network analysis of significantly dysregulated proteins was conducted to investigate the molecular processes associated with diabetes. This pinpointed ERK1/2 signaling as a key regulatory node in the diabetes-induced pathophysiology for both tissue types. The detrusor muscle samples showed diabetes-induced increased tissue remodeling-type events such as Actin Cytoskeleton Signaling and Signaling by Rho Family GTPases. The diabetic urothelium samples exhibited oxidative stress responses, as seen in the suppression of protein expression for key players in the NRF2-Mediated Oxidative Stress Response pathway. These results suggest that diabetes induced elevated inflammatory responses, oxidative stress, and tissue remodeling are involved in the development of tissue specific diabetic bladder dysfunctions. Validation of signaling dysregulation as a function of diabetes was performed using Western blotting. These data illustrated changes in ERK1/2 phosphorylation as a function of diabetes, with significant decreases in diabetes-associated phosphorylation in urothelium, but the opposite effect in detrusor muscle. These data highlight the importance of understanding tissue specific effects of disease process in understanding pathophysiology in complex disease and pave the way for future studies to better understand important molecular targets in reversing bladder dysfunction. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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