Xiong Shu1, Kai-Yue Cao2, Hui-Qi Liu3, Long Yu4, Li-Xin Sun4, Zhi-Hua Yang4, Cheng-Ai Wu5, Yu-Liang Ran6. 1. Laboratory of Molecular Orthopaedics, Beijing Research Institute of Orthopaedics and Traumatology, Beijing JiShuiTan Hospital, No. 31 Xinjiekou E Road, Xicheng, Beijing, 100035, People's Republic of China. 2. Department of Pathology, Tianjin First Central Hospital, Tianjin, People's Republic of China. 3. Department of Basic Medical Science, Medical School of Qinghai University, Xining, People's Republic of China. 4. State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 17 Panjiayuan Subdistrict, Chaoyang, Beijing, 100021, People's Republic of China. 5. Laboratory of Molecular Orthopaedics, Beijing Research Institute of Orthopaedics and Traumatology, Beijing JiShuiTan Hospital, No. 31 Xinjiekou E Road, Xicheng, Beijing, 100035, People's Republic of China. wuchengai05@163.com. 6. State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 17 Panjiayuan Subdistrict, Chaoyang, Beijing, 100021, People's Republic of China. ran_yuliang@126.com.
Abstract
BACKGROUND: Tumor-associated antigens (TAAs) can be targeted in cancer therapy. We previously identified a monoclonal antibody (mAb) 12C7, which presented anti-tumor activity in lung cancer stem cells (LCSCs). Here, we aimed to identify the target antigen for 12C7 and confirm its role in LCSCs. METHODS: Immunofluorescence was used for antigen localization. After targeted antigen purification by electrophoresis and immunoblot, the antigen was identified by LC-MALDI-TOF/TOF mass spectrometry, immunofluorescence, and immunoprecipitation. The overexpression or silence of ENO1 was induced by lentiviral transduction. Self-renewal, growth, and invasion of LCSCs were evaluated by sphere formation, colony formation, and invasion assay, respectively. High-throughput transcriptome sequencing (RNA-seq) and bioinformatics analysis were performed to analyze downstream targets and pathways of targeted antigen. RESULTS: Targeted antigen showed a surface antigen expression pattern, and the 43-55 kDa protein band was identified as α-enolase (ENO1). Self-renewal, growth, and invasion abilities of LCSCs were remarkably inhibited by ENO1 downregulation, while enhanced by ENO1 upregulation. RNA-seq and bioinformatics analysis eventually screened 4 self-renewal-related and 6 invasion-related differentially expressed genes. GSEA analysis and qRT-PCR verified that ENO1 regulated self-renewal, invasion-related genes, and pathways. KEGG pathway analysis and immunoblot demonstrated that ENO1 inactivated AMPK pathway and activated mTOR pathway in LCSCs. CONCLUSIONS: ENO1 is identified as a targeted antigen of mAb 12C7 and plays a pivotal role in facilitating self-renewal, growth, and invasion of LCSCs. These findings provide a potent therapeutic target for the stem cell therapy for lung cancer and have potential to improve the anti-tumor activity of 12C7.
BACKGROUND:Tumor-associated antigens (TAAs) can be targeted in cancer therapy. We previously identified a monoclonal antibody (mAb) 12C7, which presented anti-tumor activity in lung cancer stem cells (LCSCs). Here, we aimed to identify the target antigen for 12C7 and confirm its role in LCSCs. METHODS: Immunofluorescence was used for antigen localization. After targeted antigen purification by electrophoresis and immunoblot, the antigen was identified by LC-MALDI-TOF/TOF mass spectrometry, immunofluorescence, and immunoprecipitation. The overexpression or silence of ENO1 was induced by lentiviral transduction. Self-renewal, growth, and invasion of LCSCs were evaluated by sphere formation, colony formation, and invasion assay, respectively. High-throughput transcriptome sequencing (RNA-seq) and bioinformatics analysis were performed to analyze downstream targets and pathways of targeted antigen. RESULTS: Targeted antigen showed a surface antigen expression pattern, and the 43-55 kDa protein band was identified as α-enolase (ENO1). Self-renewal, growth, and invasion abilities of LCSCs were remarkably inhibited by ENO1 downregulation, while enhanced by ENO1 upregulation. RNA-seq and bioinformatics analysis eventually screened 4 self-renewal-related and 6 invasion-related differentially expressed genes. GSEA analysis and qRT-PCR verified that ENO1 regulated self-renewal, invasion-related genes, and pathways. KEGG pathway analysis and immunoblot demonstrated that ENO1 inactivated AMPK pathway and activated mTOR pathway in LCSCs. CONCLUSIONS:ENO1 is identified as a targeted antigen of mAb 12C7 and plays a pivotal role in facilitating self-renewal, growth, and invasion of LCSCs. These findings provide a potent therapeutic target for the stem cell therapy for lung cancer and have potential to improve the anti-tumor activity of 12C7.
Authors: K Imai; S Hirata; A Irie; S Senju; Y Ikuta; K Yokomine; M Harao; M Inoue; Y Tomita; T Tsunoda; H Nakagawa; Y Nakamura; H Baba; Y Nishimura Journal: Br J Cancer Date: 2010-12-21 Impact factor: 7.640