Primary data_F Mundt et al_Syndecan-1 as a biomarker_2014

Dear reader, this Excel file accompanies and regards the article: Diagnostic and prognostic value of soluble syndecan-1 for pleural malignancies. F. Mundt et al., 2013. In the file you will find univariate expression levels of syndecan-1 and osteopontin. Patient groups and fluid (pleural effusions or sera) are clearly labelled

Novel Genes and Pathways Modulated by Syndecan-1: Implications for the Proliferation and Cell-Cycle Regulation of Malignant Mesothelioma Cells

<div><p>Malignant pleural mesothelioma is a highly malignant tumor, originating from mesothelial cells of the serous cavities. In mesothelioma the expression of syndecan-1 correlates to epithelioid morphology and inhibition of growth and migration. Our previous data suggest a complex role of syndecan-1 in mesothelioma cell proliferation although the exact underlying molecular mechanisms are not completely elucidated. The aim of this study is therefore to disclose critical genes and pathways affected by syndecan-1 in mesothelioma; in order to better understand its importance for tumor cell growth and proliferation. We modulated the expression of syndecan-1 in a human mesothelioma cell line via both overexpression and silencing, and followed the transcriptomic responses with microarray analysis. To project the transcriptome analysis on the full-dimensional picture of cellular regulation, we applied pathway analysis using Ingenuity Pathway Analysis (IPA) and a novel method of network enrichment analysis (NEA) which elucidated signaling relations between differentially expressed genes and pathways acting via various molecular mechanisms. Syndecan-1 overexpression had profound effects on genes involved in regulation of cell growth, cell cycle progression, adhesion, migration and extracellular matrix organization. In particular, expression of several growth factors, interleukins, and enzymes of importance for heparan sulfate sulfation pattern, extracellular matrix proteins and proteoglycans were significantly altered. Syndecan-1 silencing had less powerful effect on the transcriptome compared to overexpression, which can be explained by the already low initial syndecan-1 level of these cells. Nevertheless, 14 genes showed response to both up- and downregulation of syndecan-1. The “cytokine – cytokine-receptor interaction”, the TGF-β, EGF, VEGF and ERK/MAPK pathways were enriched in both experimental settings. Most strikingly, nearly all analyzed pathways related to cell cycle were enriched after syndecan-1 silencing and depleted after syndecan-1 overexpression. Syndecan-1 regulates proliferation in a highly complex way, although the exact contribution of the altered pathways necessitates further functional studies.</p> </div

Effect of syndecan-1 silencing on cell growth (A) doubling time (B), cell-cycle distribution (C) and apoptosis (D).

<p>(<b>A</b>) Proliferation of cells silenced for syndecan-1 and corresponding scrambled control was measured using WST1 proliferation assay. Values are mean of cell number ± SD (n = 3), obtained from three independent experiments with 4 replicates in each. (<b>B</b>) Doubling time was calculated from the logarithmic phase of the growth curve. Silencing of syndecan-1 significantly decreased proliferation of mesothelioma cells (*p<0.05, ***p<0.001). (<b>C</b>) Cell cycle analysis was performed by propidium iodide staining followed by flow cytometry, 24 and 48 hours after silencing. Columns represents mean percentage of cells in each phase of the cell cycle ± SD (n = 3) (<b>D</b>) Apoptosis was measured by flow cytometry using Annexin-V-FITC and Propidium iodide (PI) staining. Cells silenced for syndecan-1 and scrambled controls were stained with PI and Annexin-V, 24 and 48 hours after syndecan-1 silencing. Three independent experiments were performed. Representative Annexin V/PI plots are shown where X axis shows the log of fluorescence intensity of Annexin-V and Y axis demonstrate the log of fluorescence intensity of PI. Cells in the lower left quadrant represent living cells, in the lower right quadrant early apoptotic cells; the upper right and left quadrant show late apoptotic and necrotic cells, respectively. No significant change in apoptosis was recorded. Asterisks indicate statistically significant differences compared to the scrambled control (* p≤0.05, ** p≤0.01, *** p≤0.001). A two-tailed t test was performed to test the statistical significance between cells silenced for syndecan-1 and scrambled control. SD = standard deviation.</p

Networks related to proliferation (A) and cell-cycle regulation (B) according to Ingenuity Pathway (IPA).

<p>Differentially expressed genes which by functional analysis were associated to (<b>A</b>) proliferation or (<b>B</b>) cell cycle analysis were uploaded into IPA. Genes were overlaid onto a global molecular network developed from information contained in the Ingenuity® Knowledge Base. The obtained networks were algorithmically generated based on the connectivity of differentially expressed genes. The networks also reveal the relationship between different genes and their subcellular distribution according to IPA score from published data. Red symbols denote upregulated genes and green symbols denote downregulated genes.</p

Validation of syndecan-1 silencing (A) and microarray data (B–D).

<p>(<b>A</b>) Succesful silencing of syndecan-1 was confirmed by RT-PCR and flow cytometry. The level of syndecan-1 mRNA (left column) and protein (right column) after silencing compared to cells treated with negative/scrambled control, 24 hours after silencing. The silencing is highly significant (p<0.0001 for mRNA and p<0.01 for protein ) as calculated by a one-sided t-test (n = 3). Selected transcripts deregulated in microarray were analyzed by RT-PCR for syndecan-1 overexpression (<b>B</b>) and silencing (<b>C</b>) and/or by proteome profile array (<b>D</b>) for syndecan-1 overexpression. mRNA or protein level of cells with modulated syndecan-1 was compared to their specific control (vector for overexpressed and negative-scrambled for silenced). Results are given in fold changes (FC). Light gray columns represent the values obtained by microarray, dark gray columns correspond to the results from RT-PCR or from proteome profiler array. Each value represents an average of fold-changes of three independent experiments, error bars represent standard deviation (SD); statistically significant changes: *p<0.05, ** p<0.01, **** p<0.0001 for differential expression of transcripts in syndecan-1 modulated cells compared to the corresponding controls.</p

Genes affected by both syndecan-1 overexpression and silencing.

<p>FC_overexp represents fold changes for expression levels following syndecan-1 upregulation compared to vector transfected control cells. FC_silenced represents fold changes after silencing of syndecan-1 compared to control cells treated with negative/scrambled siRNA. All gene changes are significant at q≤0.05. Fold changes and q-values were calculated using OCplus package in R.</p

Differentially expressed genes following syndecan-1 overexpression grouped by functional categories according to Gene Ontology.

<p>Each differentially expressed gene (FC≥1.5 or FC ≤−1.5, q≤0.05 using OCplus test from R package) was assigned to functional categories using Gene Ontology database. Genes from each category were counted and plotted on the graph. Vertical axis represents the number of differentially expressed genes and each column represents a GO category.</p

Common pathways significantly affected by both syndecan-1 overexpression and silencing, revealed by Ingenuity Pathway (IPA).

<p>Canonical pathway analysis identified the pathways from the IPA library that were most significant to the data set. Molecules from the data set that met the cutoff of FC≥1.5 or FC≤−1.5 and q≤0.05 and were associated with a canonical pathway in the Ingenuity Knowledge Base were considered for the analysis. The significance of the association between the data set and the canonical pathway was measured by Fisher’s exact test. Grey columns denote syndecan-1 overexpression and black columns denote syndecan-1 silencing. Bars represent the logarithmic values of the significance level (p), the dashed line represents the threshold of p = 0.05.</p

Most significantly altered cellular and molecular functions revealed by Ingenuity Pathway Analysis (IPA).

<p>(<b>A</b>) Functions significantly associated to syndecan-1 overexpression or silencing. Light gray bars show the gene set for syndecan-1 overexpression, dark gray bars show the gene set for syndecan-1 silencing. (<b>B</b>) Functions altered concomitantly by both overexpression and silencing. Molecules from both datasets that met the FC≥1.5 or FC≤−1.5 and q≤0.05 cutoff criteria were considered for the analysis. Right-tailed Fisher’s exact test was used to calculate a p-value. Bars represent the logarithmic values of the significance level (p), the dashed line corresponds to the threshold of p = 0.05.</p

Pathways influenced by both syndecan-1 overexpression and silencing, revealed by network enrichment (NEA) analysis.

<p>Altered KEGG pathways (<b>A</b>); pathways determining signaling and growth factor activity (<b>B</b>); pathways related to particular phases of the cell cycle (<b>C</b>). Diamonds represent differentially expressed genes (E2FL denotes cells transfected with full-length syndecan-1 vs. cells transfected with empty vector; NS2SI denotes cells silenced for syndecan-1vs scrambled control); circles represent pathways from different databases (FGS). Red lines denote enriched pathways, blue lines denote depleted pathways. Line width is proportional with the number of individual gene<b>-</b>gene network links between two gene sets (linear scales from <i>min</i> 7 to <i>max</i> 3,995). Line opacity represents confidence (FDR<0.1 for every shown link). Numbers at lines correspond to Z-scores from network enrichment analysis of differentially expressed genes as groups versus genes of FGS as groups. The picture was generated using the stand-alone network software Cytoscape <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048091#pone.0048091-Shannon1" target="_blank">[109]</a> using network edges and nodes from the custom NEA software.</p