4378 ONCOLOGY LETTERS 13: 4378-4384, 2017 Identification of candidate drugs for the treatment of metastatic osteosarcoma through a subpathway analysis method XIN LI, MING-LAN YAN and QIAN YU Department of Pharmacy, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China Received October 8, 2015; Accepted December 6, 2016 DOI: 10.3892/ol.2017.5953 Abstract. Osteosarcoma (OS) is the third most frequent type of adolescent growth spurt indicates there is an association between cancer in adolescents and represents >56% of all bone tumors. this disease and bone development (4,5). The introduction of In addition, metastatic OS frequently demonstrates resistance preoperative high-dose combined chemotherapy in the last to conventional chemotherapy; thus, the development of novel three decades has significantly improved the disease‑free 5‑year therapeutic agents for the treatment of patients with metastatic survival rate of young patients (<40 years old) to ~50% (6). OS is warranted. In the present study, the metabolic mechanisms However, OS is highly aggressive and numerous patients with underlying OS metastasis were investigated using a subpathway OS develop metastases, primarily in the lung, even following analysis method and lead to the identification of candidate resection of the primary tumor (2,7). Furthermore, metastatic OS drugs for the treatment of metastatic OS. Using the GSE14827 frequently exhibits resistance to conventional chemotherapies microarray dataset from the Gene Expression Omnibus data- that were effective for treatment of the primary tumor and >30% base, 546 differentially expressed genes were identified between of metastatic OS cases do not respond to chemotherapy (4,8,9). samples from patients with OS who did or did not develop The chemoresistance of malignant OS limits the effectiveness metastatic OS. Furthermore, nine significantly enriched meta- of current cytotoxic drugs (10). Therefore, elucidation of the bolic subpathways were identified, which may be involved in OS mechanisms underlying the metastasis of OS and the develop- metastasis. Finally, using an integrated analysis of metastatic ment of novel drugs to overcome chemoresistance in this OS-associated subpathways and drug-affected subpathways, disease are warranted to improve the survival rate of patients 98 small molecule drug candidates capable of targeting the meta- with OS (1). static OS‑associated subpathways were identified. This method The development of novel drugs is a time-consuming and identified existing anti‑cancer drugs, including semustine, in labor-intensive process. Drug repositioning, which explores addition to predicting potential drugs, such as lansoprazole, for potential novel uses for known drugs, has become an effec- the treatment of metastatic OS. Transwell and wound healing tive and innovative approach to the drug development process, assays demonstrated that lansoprazole reduced the invasiveness particularly with the development of system biology and avail- and migration of U2OS cells. These small molecule drug candi- ability of biochemical information in public databases (10). dates identified through a bioinformatics approach may provide For example, the Gene Expression Omnibus (GEO) database insights into novel therapy options for the treatment of patients and the Connectivity Map (CMap) database have provided with metastatic OS. numerous microarray datasets under disease or drug-induced conditions (11). Introduction In the present study, a bioinformatics method based on meta- bolic subpathway analysis was used to identify potential drugs Osteosarcoma (OS) is the third most frequent type of cancer for the treatment of metastatic OS. Differentially expressed in adolescents and represents >56% of all bone tumors (1). The genes (DEGs) between patients with OS who relapsed and median age of patients with OS is 16 years old, with a male those who did not were identified. In addition, existing small predominance (2,3). The high incidence of OS during the molecule drugs capable of targeting metabolic subpathways associated with OS metastasis were considered as potential novel agents for the treatment of metastatic OS. Furthermore, it was experimentally verified that lansoprazole could inhibit the invasion of U2OS cells. The candidate drugs identified by the Correspondence to: Dr Qian Yu, Department of Pharmacy, approach used in the current study may improve the survival of China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun, Jilin 130033, P.R. China patients with metastatic OS in the future. E-mail: [email protected] Materials and methods Key words: metabolic subpathways, network, drugs, metastatic osteosarcoma Microarray data and DEG analysis. The microarray dataset GSE14827 was downloaded from the GEO database (National Center of Biotechnology Information, Bethesda, MD, USA; LI et al: IDENTIFICATION OF CANDIDATE DRUGS FOR METASTATIC OSTEOSARCOMA 4379 www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14827). between OS metastasis and the drugs were obtained for use in The dataset included biopsy samples from 9 patients with OS the present study. Among the 403 subpathways, 9 subpathways who developed pulmonary metastases ≤4 years following neoad- corresponding to 98 drugs were associated with OS metastasis. juvant chemotherapy and curative resection, and 18 patients Detailed information and Anatomical Therapeutic Chemical who did not relapse in this time frame (7). All tumor samples in classification for these drugs were obtained from the Drug Bank the dataset were obtained through diagnostic incisional biopsies database (www.drugbank.ca). from primary sites of OS prior to neoadjuvant chemotherapy at the National Cancer Center Hospital (Tokyo, Japan) between Transwell invasion assay. U2OS cells (obtained from the March 1996 and September 2007 (7). American Type Culture Collection, Manassas, VA, USA) were Raw microarray data and probe annotation files from the starved through culturing in serum-free medium [RPMI-1640 dataset were downloaded for analysis. The data downloaded (MGC‑803); Gibco; Fisher Scientific, Inc., Waltham, MA, USA] included the expression levels of 54,613 probe sets across for 12 h under standard conditions, as described by the American 27 samples from patients with OS (age, 8-38 years; 13 males Type Culture Collection. The cells were subsequently seeded and 14 females) that were analyzed using the GeneChip® into 6-well plates at a density of 2.5x105 cells/well. Following Human Genome U133 Plus 2.0 array (Affymetrix, Inc., Santa treatment with 100 µmol lansoprazole for 24 h at 37˚C, cells Clara, CA, USA). In the present study, the fold change values were fixed and stained and their invasiveness was investigated for each probe were determined. The fold change value for using a Transwell chamber (Corning Incorporated, Corning, each probe is the average expression value of OS samples NY, USA). BD Matrigel™ Basement Membrane Matrix divided by that of normal samples. Subsequently, each probe (50 mg/l; BD Biosciences, San Jose, CA, USA) was diluted was converted into an Entrez Gene ID (www.ncbi.nlm.nih.gov/ with serum-free medium at a ratio of 1:8, and each Transwell gene). If a gene mapped onto >1 probe, the mean expression chamber was coated with 60 µl of this solution. Prior to use, value of all corresponding probes was considered the expres- the polycarbonate membrane (pore size, 8 µm) was hydrated sion value of the gene. Genes with a fold change value of >1.5 with 50 µl of serum-free medium containing 10 g/l bovine or <0.667 were identified as DEGs. serum albumin (Beyotime Institute of Biotechnology, Haimen, China) at 37˚C for 30 min. Treated cells (8x105cells/well) Identification of subpathways associated with OS metastasis. were subsequently seeded into the upper chamber in 200 µl of The Subpathway Miner R package (version 1.0; cran.r-project. serum-free medium, and 600 µl of medium containing 10% org/src/contrib/Archive/SubpathwayMiner), which is a flexible fetal bovine serum (Stemcell Technologies, Inc., Vancouver, subpathway identification software, was used to obtain subpath- BC, Canada) was added to the lower chamber as an attractant. ways associated with OS metastasis (12). DEGs between patients Following incubation for 24 h at 37˚C in 5% CO2, the cells that with OS who did or did not develop metastases were imported had invaded the lower surface of the filter were fixed with 75% into Subpathway Miner, which identified significantly enriched ethanol and stained with 0.1% crystal violet. Cells were counted subpathways using hypergeometric tests (12). This software by eye in three random areas in each chamber (magnification, converts pathway structure data from the Kyoto Encyclopedia x200) using a light microscope. of Genes and Genomes (KEGG) database (www.genome.jp/ kegg) into undirected R graph objects. In the pathways, proteins Wound healing assay. The migration ability of U2OS cells are considered nodes and any two nodes belonging to the was determined using a wound healing assay. U2OS cells were same reaction are connected by an edge. Finally, Subpathway seeded into 6-well plates (2x105). When the cells reached 70% Miner divides the entire pathway into subpathways using the confluence they were treated with lansoprazole (200 µM). The ʻk-cliqueʼ method, which is defined as a sub‑graph in which control group was treated with DMSO.A sterile 10 µl pipette the distance between any two nodes is <k (12). In the present tip was used to draw straight lines