Abstract
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.
Highlights
Syndecans are a family of cell surface heparan sulfate proteoglycans (HSPGs) with an extracellular domain carrying glycosaminoglycan (GAG) side chains, a transmembrane domain and a short cytoplasmic domain [1]
The cell cycle analysis showed that due to syndecan-1 silencing the amount of G2/M cells was significantly reduced after 24 hours, while an increased number of cells were seen in G0/G1 phase in cells silenced for syndecan-1, compared to scrambled control (Figure 1C)
In order to interpret alterations generated by syndecan-1 in mesothelioma cells, we combined traditional strategies of gene expression analysis with a novel network enrichment analysis, which takes into account functional coupling in gene networks [29,34]
Summary
Syndecans are a family of cell surface heparan sulfate proteoglycans (HSPGs) with an extracellular domain carrying glycosaminoglycan (GAG) side chains, a transmembrane domain and a short cytoplasmic domain [1]. A number of studies have shown that syndecans play critical roles in cellular processes including differentiation, cell adhesion [2,3], cytoskeletal organization, cell spreading and migration [4,5,6], infiltration, angiogenesis [7,8] and proliferation of various malignant tumors [8,9,10] Syndecans exert these functions partly through their GAG chains, mainly heparan sulfate, but recent studies show that different domains of the core protein have distinct roles as well [3,11]. In mesenchymal tumors its expression level is generally low, only few studies have addressed syndecan-1’s role and regulation in these tumors [13,14]
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