3.2. Functional Analysis of Differentially Regulated Genes
A gene ontology (GO) analysis of differentially expressed genes was performed using ToppGene Suite [16]; “response to hormone”, “extracellular matrix organization”, “cell migration”, “ossification” and “vasculature development” were identified to be a few of the most enriched biological processes. The top 200 enriched biological processes are listed in Table S3, while most relevant GO terms and associated genes are listed in Table 2. Of all the differentially expressed genes, secreted signaling proteins Tgfb2, Il-6, Cxcl1, and Ctgf and metallopeptidases Mmp13, Adamts4, and Adamts5 were of particular interest, as these genes have previously been shown to play a role in regulating cancer migration and invasion [29,30,31,32,33,34,35,36] and bone remodeling [37,38,39,40,41].
microarrays-04-00503-t002_Table 2 Table 2  Enriched gene ontology terms associated with genes differentially expressed between UMR cells co-cultured with PC3 cells and UMR monocultures.   To better understand how secreted signaling proteins Tgfb2, Il-6, Cxcl1, and Ctgf may be involved in potentially regulating a complex biological process such as bone metastasis, we examined these genes in the context of protein interaction networks. GeneMANIA [17] and Cytoscape [18] were used to generate and visualize protein interactions between these genes and related genes in the network. This interaction data included physical and predicted protein–protein interactions. An integrated network of Tgfb2, Il-6, Cxcl1, and Ctgf interactions revealed several putative binding partners in osteoblasts and/or prostate cancer and suggested that these secreted cytokines may control up-regulated transcription factors Junb, Cebpb and Stat3 in osteoblasts (Figure 2). These transcription factors have been shown to play a major role in regulating bone metabolism [42,43,44,45]. This analysis also revealed several other up-regulated genes such as A2m and Nfkbiz as members of Il-6, Cxcl1, Ctgf, and Tgfb2 interactome (Figure 2). Future experimental studies may validate the potential role up-regulation of Il-6, Cxcl1, Ctgf, and Tgfb2 may have in promoting cancer cell migration, invasion, and cancer-induced bone metabolism.
Figure 2  An integrated network of Tgfb2, Il-6, Cxcl1, and Ctgf interactions generated using GeneMANIA and Cytoscape. Query genes (triangles) and up-regulated genes (diamonds) are highlighted.   Metallopeptidases Mmp13, Adamts4, and Adamts5 are associated with the enriched gene ontology term “extra cellular matrix organization”. These metallopeptidases regulate bone remodeling by degrading components of the extracellular matrix, particularly the collagens and aggrecans [36,41,46]. We found Mmp13 to increase greater than 60 fold in osteoblast co-cultures compared to monocultures (Figure 1C). Mmp13 has also been shown to regulate cancer-induced osteolysis [47,48,49]. Adamts4 and Adamts5 have been shown to promote cell growth and invasion [36]. We also identified another up-regulated Adam family member, Adamts1, in osteoblast co-cultures and confirmed the differential regulation of this transcript by qPCR (Figure 1C). Up-regulation of these metallopeptidases may enhance bone remodeling and promote prostate cancer invasion and growth [36,48].