Systems biology approach reveals a link between mTORC1 and G2/M DNA damage checkpoint recovery.
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ABSTRACT:
SUBMITTER: Hsieh HJ
PROVIDER: S-EPMC6162282 | biostudies-literature | 2018 Sep
REPOSITORIES: biostudies-literature
Publications
Systems biology approach reveals a link between mTORC1 and G2/M DNA damage checkpoint recovery.
Hsieh Hui-Ju HJ Zhang Wei W Lin Shu-Hong SH Yang Wen-Hao WH Wang Jun-Zhong JZ Shen Jianfeng J Zhang Yiran Y Lu Yiling Y Wang Hua H Yu Jane J Mills Gordon B GB Peng Guang G
Nature communications 20180928 1
Checkpoint recovery, the process that checkpoint-arrested cells with normal DNA repair capacity resume cell cycle progression, is essential for genome stability. However, the signaling network of the process has not been clearly defined. Here, we combine functional proteomics, mathematical modeling, and molecular biology to identify mTORC1, the nutrient signaling integrator, as the determinant for G2/M checkpoint recovery. Inhibition of the mTORC1 pathway delays mitotic entry after DNA damage th ...[more]
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Project description:Comparative phosphoproteomic analysis of checkpoint recovery identifies new regulators of the DNA damage response. How cells recover from a DNA damage-induced arrest is currently poorly understood. We have performed large-scale quantitative phosphoproteomics to identify changes in protein phosphorylation that occur during recovery from a G2 arrest. We identified 154 proteins that were differentially phosphorylated in multiple experiments. Systematic depletion of each of these differentially phosphorylated proteins by siRNA identified at least 10 potential regulators of recovery. Data processing and bioinformatics: Raw data were converted to single DTA files using DTA SuperCharge and merged into Mascot Generic Format (MGF) files, which were searched using an in-house licensed Mascot v2.2 search engine against Human Swissprot 56.2 database concatenated with reversed sequences as decoy (containing 40656 sequences, 20328 forward sequences). Carbamidomethyl cysteine and oxidized methionines were set as fixed modifications; serine, threonine, and tyrosine phosphorylations were set as variable modifications; quantification was set with dimethyl double mode. The mass tolerance of the precursor ion was 10 ppm and 0.9 Da for fragment ions. The false discovery rate (FDR) was determined as < 1% (Mascot score threshold of 31) using the decoy database approach and the MGF files were trimmed at Mascot score threshold of 31 (1% FDR) by RockerBox. MSQuant v1.5 was used to quantitate the amounts of the identified phosphopeptides and determine the exact phosphorylation site within the peptide. Every phosphopeptide quantitation was manually validated; peptides with low signal:noise ratios, low number of MS scans, or overlapping peaks were not included for quantitative purposes. Histogram plots of the ratio of whole quantified peptides in each experiment were used to normalize the ratios. A program to extract information from MSQuant output was developed in-house to identify the position of each phosphorylation site and its status in current available databases (Phospho ELM and SwissProt). Panther Classification System was used to classify proteins with regulated phosphopeptides. NetworKIN was used to predict in vivo kinases for the phosphosites.
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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.
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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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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
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