ABSTRACT
Synovial sarcoma (SS) is a rare high-grade malignant mesenchymal tumour with a relatively poor prognosis despite intensive multimodal therapy. Although pazopanib, a multi-kinase inhibitor, is often used for advanced SS, most cases eventually become resistant to pazopanib. In the present study, we investigated the mechanisms of acquired pazopanib resistance in SS. To examine acquired pazopanib resistance, two SS cell lines, SYO-1 and HS-SY-II, were isolated after multiple selection steps with increasing concentrations of pazopanib. SYO-1 was also used in vivo. Then, pazopanib-resistant clones were investigated to assess potential mechanisms of acquired pazopanib resistance. Stable pazopanib-resistant clones were established and exhibited enhanced cell cycle progression, cell growth with increased ERK1/2 phosphorylation, and higher sensitivity than parental cells to a MEK-inhibitor, trametinib, both in vitro and in vivo. Furthermore, addition of low-dose trametinib partially reversed the pazopanib resistance. In the pazopanib-resistant clones, dual specificity phosphatase 6 (DUSP6) was downregulated. Inhibition of DUSP6 expression in parental HS-SY-II cells partially recapitulated acquired pazopanib resistance. Acquired pazopanib resistance in SS was associated with activation of ERK1/2 through downregulation of DUSP6 expression. Simultaneous treatment with pazopanib and a MEK inhibitor could be a promising strategy to overcome pazopanib resistance in SS.
Subject(s)
Dual Specificity Phosphatase 6/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Sulfonamides/pharmacology , Animals , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation/drug effects , Drug Resistance, Neoplasm/drug effects , Dual Specificity Phosphatase 6/antagonists & inhibitors , Female , Humans , Indazoles , Mice , Mice, Inbred BALB C , Mice, Nude , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-raf/genetics , Pyridones/pharmacology , Pyrimidines/therapeutic use , Pyrimidinones/pharmacology , Sarcoma, Synovial/drug therapy , Sarcoma, Synovial/metabolism , Sarcoma, Synovial/pathology , Sulfonamides/therapeutic use , ras Proteins/geneticsABSTRACT
Ewing sarcoma (ES) is a small round-cell tumor of the bones and soft tissues. ES frequently causes distant metastases, particularly in the lung and bone, which worsens patient prognosis. Cadherin-11 (Cad-11) is an adhesion molecule that is highly expressed in osteoblasts. Its expression is associated with bone metastases in prostate and breast cancer patients, and is known to occur in ES. Here we investigated the effects of Cad-11 on bone metastases of ES. Human ES cell lines RD-ES, SK-ES-1, SK-N-MC, and TC-71 cells were transduced with lentivirus containing Cad-11 shRNA or control shRNA (ES/Cad-11 and ES/Ctr). RD-ES and TC-71 were infected with a lentivirus luciferase vector. Adhesion assays were performed using these cells and recombinant Cad-11-Fc chimera or mouse osteoblast cell line MC3T3-E1. Cell motility was investigated via wound-healing assay. Intracardiac injection of RD-ES/Cad-11 and RD-ES/Ctr was used to create a mouse model of experimental bone metastasis. The association between Cad-11 expression and bone metastases and clinical prognosis in ES patients was analyzed by immunohistochemistry. We found knockdown of Cad-11 in ES cells resulted in reduced attachment ability and cell motility. In a mouse model of metastasis, RD-ES/Cad-11 cells caused fewer metastases than RD-ES/Ctr cells. The expression of Cad-11 in ES patients was significantly related to bone metastases (P < 0.05, logistic regression) and poorer overall survival (P < 0.05, log-rank test). These findings may explain that Cad-11 in ES cells may be essential for cell adhesion and motility, and is a promising molecular target for patients with ES.
Subject(s)
Antibodies, Monoclonal/pharmacology , Bone Neoplasms/secondary , Cadherins/metabolism , Cell Movement , Osteoblasts/pathology , Sarcoma, Ewing/pathology , Animals , Apoptosis , Blotting, Western , Bone Neoplasms/metabolism , Bone Neoplasms/mortality , Cadherins/antagonists & inhibitors , Cadherins/immunology , Cell Adhesion , Cell Proliferation , Coculture Techniques , Female , Flow Cytometry , Humans , Immunoenzyme Techniques , Luciferases/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Staging , Osteoblasts/metabolism , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/mortality , Survival Rate , Tumor Cells, Cultured , Wound Healing , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Diverse functions of microRNAs (miRNAs), including effects on tumorigenesis, proliferation, and differentiation, have been reported, and several miRNAs have also been demonstrated to play an important role in apoptosis. In this study, we investigated the possible role that miRNAs may play in the development of chemoresistance in Ewing sarcoma/primitive neuroectodermal tumor (EWS). METHODS: We screened doxorubicin (Dox)-resistant EWS cells to identify any distinct miRNA sequences that may regulate the chemoresistance of EWS cells. The effects of miRNAs were evaluated using a chemosensitivity assay. The possible target genes of the miRNAs were predicted using a web-based prediction program. RESULTS: We found miR-125b to be upregulated in two different Dox-resistant EWS cell lines. The upregulation of miR-125b was also confirmed in the EWS tumors having survived chemotherapy regimen which includes doxorubicin. When miR-125b was knocked down in EWS cells, both the Dox-resistant and parental cells showed an enhanced sensitivity to doxorubicin, which was associated with the upregulation of the pro-apoptotic molecules, p53 and Bak. Inversely, the overexpression of miR-125b in parental EWS cells resulted in enhanced drug resistance, not only to doxorubicin, but also to etoposide and vincristine. CONCLUSIONS: Our findings suggest that miR-125b may play a role in the development of chemoresistance in EWS by suppressing the expression of the apoptotic mediators, such as p53 and Bak.
