Project description:Background Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. The survival rate of patients with metastatic disease remains very dismal. Nevertheless, metastasis is a complex process and a single-level analysis is not likely to identify its key biological determinants. In this study, we used a systems biology approach to identify common metastatic pathways that are jointly supported by both mRNA and protein expression data in two distinct human metastatic OS models. Results mRNA expression microarray and N-linked glycoproteomic analyses were performed on two commonly used isogenic pairs of human metastatic OS cell lines, namely HOS/143B and SaOS-2/LM7. Pathway analysis of the differentially regulated genes and glycoproteins separately revealed pathways associated to metastasis including cell cycle regulation, immune response, and epithelial-to-mesenchymal-transition. However, no common significant pathway was found at both genomic and proteomic levels between the two metastatic models, suggesting a very different biological nature of the cell lines. To address this issue, we used a topological significance analysis based on a “shortest path” algorithm to identify topological nodes, which uncovered additional biological information with respect to the genomic and glycoproteomic profiles but remained hidden from the direct analyses. Pathway analysis of the significant topological nodes revealed a striking concordance between the models and identified significant common pathways, including “Cytoskeleton remodeling/TGF/WNT”, “Cytoskeleton remodeling/Cytoskeleton remodeling”, and “Cell adhesion/Chemokines and adhesion”. Of these, the “Cytoskeleton remodeling/TGF/WNT” was the top ranked common pathway from the topological analysis of the genomic and proteomic profiles in the two metastatic models. The up-regulation of proteins in the “Cytoskeleton remodeling/TGF/WNT” pathway in the SaOS-2/LM7 and HOS/143B models was further validated using an orthogonal Reverse Phase Protein Array platform. Conclusions In this study, we used a systems biology approach by integrating genomic and proteomic data to identify key and common metastatic mechanisms in OS. The use of the topological analysis revealed hidden biological pathways that are known to play critical roles in metastasis. Wnt signaling has been previously implicated in OS and other tumors, and inhibitors of Wnt signaling pathways are available for clinical testing. Further characterization of this common pathway and other topological pathways identified from this study may lead to a novel therapeutic strategy for the treatment of metastatic OS.

Project description:Since the worldwide increase in obesity represents a growing challenge for the health care systems, new approaches are needed to treat obesity and associated disease effectively. The food intake is primarily stored in the adipose tissue and therefore this organ is in focus to develop new anti-obesity treatments. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. Beside well known regulators of adipogenesis and neutral lipid storage (like PPAR?, RXR, Perilipin A) the screening revealed a large number of genes which were not previously described in the context of fatty tissue biology. An enrichment of genes was observed for axonemal dyneins. Five out of ten anxonemal dyneins were identified in our primary screen and retested positive with independent siRNAs and cell-donors. Quantitative RT-PCR- and immunoblot analysis revealed that axonemal dyneins are expressed in preadipocytes and maturing adipocytes. Using microarray analysis, we further characterize the remaining genes identified in our primary screen to determine their expression pattern during adipogenesis. In the course of fat cell differentiation, 149 among the 459 positive genes were regulated on the gene expression level. Assuming that an adipogenesis-specific expression pattern is another independent hind, that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we retested this gene-pool with independent siRNAs and cell donors. Finally, to show that the genes identified are per se druggable we performed a proof of principle experiment using an antagonist for one randomly chosen gene. The results showed a very similar phenotype compared to knock-down experiments proofing the druggability. Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point, to identify anti-obesity compounds targeting the adipose tissue. For microarray analysis, RNA was isolated at 3 different points in time during adipogenesis (day 0, day 3 and day 7 after induction of differentiation). This experiment was carried out with cells obtained from three different donors. No replicates are included for each point in time.

Project description:Since the worldwide increase in obesity represents a growing challenge for the health care systems, new approaches are needed to treat obesity and associated disease effectively. The food intake is primarily stored in the adipose tissue and therefore this organ is in focus to develop new anti-obesity treatments. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. Beside well known regulators of adipogenesis and neutral lipid storage (like PPAR?, RXR, Perilipin A) the screening revealed a large number of genes which were not previously described in the context of fatty tissue biology. An enrichment of genes was observed for axonemal dyneins. Five out of ten anxonemal dyneins were identified in our primary screen and retested positive with independent siRNAs and cell-donors. Quantitative RT-PCR- and immunoblot analysis revealed that axonemal dyneins are expressed in preadipocytes and maturing adipocytes. Using microarray analysis, we further characterize the remaining genes identified in our primary screen to determine their expression pattern during adipogenesis. In the course of fat cell differentiation, 149 among the 459 positive genes were regulated on the gene expression level. Assuming that an adipogenesis-specific expression pattern is another independent hind, that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we retested this gene-pool with independent siRNAs and cell donors. Finally, to show that the genes identified are per se druggable we performed a proof of principle experiment using an antagonist for one randomly chosen gene. The results showed a very similar phenotype compared to knock-down experiments proofing the druggability. Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point, to identify anti-obesity compounds targeting the adipose tissue.

Project description:Background Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. The survival rate of patients with metastatic disease remains very dismal. Nevertheless, metastasis is a complex process and a single-level analysis is not likely to identify its key biological determinants. In this study, we used a systems biology approach to identify common metastatic pathways that are jointly supported by both mRNA and protein expression data in two distinct human metastatic OS models. Results mRNA expression microarray and N-linked glycoproteomic analyses were performed on two commonly used isogenic pairs of human metastatic OS cell lines, namely HOS/143B and SaOS-2/LM7. Pathway analysis of the differentially regulated genes and glycoproteins separately revealed pathways associated to metastasis including cell cycle regulation, immune response, and epithelial-to-mesenchymal-transition. However, no common significant pathway was found at both genomic and proteomic levels between the two metastatic models, suggesting a very different biological nature of the cell lines. To address this issue, we used a topological significance analysis based on a “shortest path” algorithm to identify topological nodes, which uncovered additional biological information with respect to the genomic and glycoproteomic profiles but remained hidden from the direct analyses. Pathway analysis of the significant topological nodes revealed a striking concordance between the models and identified significant common pathways, including “Cytoskeleton remodeling/TGF/WNT”, “Cytoskeleton remodeling/Cytoskeleton remodeling”, and “Cell adhesion/Chemokines and adhesion”. Of these, the “Cytoskeleton remodeling/TGF/WNT” was the top ranked common pathway from the topological analysis of the genomic and proteomic profiles in the two metastatic models. The up-regulation of proteins in the “Cytoskeleton remodeling/TGF/WNT” pathway in the SaOS-2/LM7 and HOS/143B models was further validated using an orthogonal Reverse Phase Protein Array platform. Conclusions In this study, we used a systems biology approach by integrating genomic and proteomic data to identify key and common metastatic mechanisms in OS. The use of the topological analysis revealed hidden biological pathways that are known to play critical roles in metastasis. Wnt signaling has been previously implicated in OS and other tumors, and inhibitors of Wnt signaling pathways are available for clinical testing. Further characterization of this common pathway and other topological pathways identified from this study may lead to a novel therapeutic strategy for the treatment of metastatic OS. In this study we analyzed two human metastatic OS cell lines and their parental non-metastatic lines. The two human metastatic OS cell line models were HOS/143B and SaOS-2/LM7. The HOS cell line, originally known as M.T. and later as TE-85, was derived from an OS of a 13 year-old girl. The 143B metastatic subline was generated from HOS by a Ki-RAS oncogene transformation [Rhim, J.S., et al., Characterization of human cells transformed in vitro by N-methyl-N'-nitro-N-nitrosoguanidine. Int J Cancer, 1977. 19(4): p. 505-10.]. The SaOS-2 cell was derived from an OS of an 11 year-old girl, and its metastatic subline LM7, was developed by multiple in vivo selection of SaOS-2 cells in mice with pulmonary metastases [Jia, S.F., L.L. Worth, and E.S. Kleinerman, A nude mouse model of human osteosarcoma lung metastases for evaluating new therapeutic strategies. Clin Exp Metastasis, 1999. 17(6): p. 501-6.]. No replicates were included.

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Omics multi-layers networks and identification of the key genes and miRNAs involved in fat storage and metabolism: systems biology approach

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