Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma

et al. (2014) Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma. PLoS ONE 9(10): e109578. doi:10.1371/journal.pone.0109578 Histone Deacetylase 1/Sp1/MicroRNA-200b Signaling Accounts for Maintenance of Cancer Stem-Like Cells in Human Lung Adenocarcinoma Dong-Qin Chen 0 Jia-Yuan Huang 0 Bing Feng 0 Ban-Zhou Pan 0 Wei De 0 Rui Wang 0 Long-Bang Chen 0 Jin Q. Cheng, H.Lee Moffitt Cancer Center & Research Institute, United States of America 0 1 Department of Medical Oncology, Jinling Hospital, School of Medicine, Nanjing University , Nanjing, Jiangsu , P. R. China , 2 Department of Biochemistry and Molecular Biology, Nanjing Medical University , Nanjing , P. R. China The presence of cancer stem-like cells (CSCs) is one of the mechanisms responsible for chemoresistance that has been a major hindrance towards lung adenocarcinoma (LAD) treatment. Recently, we have identified microRNA (miR)-200b as a key regulator of chemoresistance in human docetaxel-resistant LAD cells. However, whether miR-200b has effects on regulating CSCs remains largely unclear and needs to be further elucidated. Here, we showed that miR-200b was significantly downregulated in CD133+/CD326+ cells that exhibited properties of CSCs derived from docetaxel-resistant LAD cells. Also, restoration of miR-200b could inhibit maintenance and reverse chemoresistance of CSCs. Furthermore, suppressor of zeste12 (Suz-12) was identified as a direct and functional target of miR-200b, and silencing of Suz-12 phenocopied the effects of miR-200b on CSCs. Additionally, overexpression of histone deacetylase (HDAC) 1 was identified as a pivotal mechanism responsible for miR-200b repression in CSCs through a specificity protein (Sp) 1-dependent mechanism, and restoration of miR-200b by HDAC1 repression significantly suppressed CSCs formation and reversed chemoresistance of CSCs by regulating Suz-12-E-cadherin signaling. Also, downregulation of HDAC1 or upregulation of miR-200b reduced the in vivo tumorigenicity of CSCs. Finally, Suz-12 was inversely correlated with miR-200b, positively correlated with HDAC1 and upregulated in docetaxel-resistant LAD tissues compared with docetaxel-sensitive tissues. Taken together, the HDAC1/miR200b/Suz-12-E-cadherin signaling might account for maintenance of CSCs and formation of chemoresistant phenotype in docetaxel-resistant LAD cells. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Natural Science Foundation of China (http://www.nsfc.gov.cn/) (LBC No. 81272474, BF No. 81301914). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Lung cancer accounts for the most cancer-related mortalities in both women and men worldwide [1]. Chemotherapy is an important component of the first-line therapies for lung adenocarcinoma (LAD) that constitutes the most common histological form of lung cancer. However, chemoresistance represents a predominant obstacle towards chemotherapeutic treatment of LAD, which leads to poor prognosis of the patients. Thus, exploring the possible molecular mechanisms involved in chemoresistance has become a key issue in clinical treatment of human LAD. Cancer stem-like cells (CSCs) or tumor initiating stem cells are a sub-population of tumor cells and play pivotal roles in cancer initiation, progression, recurrence and chemoresistance [25]. CSCs, derived from both CSCs and non-CSCs, give rise to tumors through self-renewal and are able to differentiate into multiple cell types [69]. Many cancer therapies including chemotherapies that kill the bulk of cancer cells, may ultimately fail as they do not eliminate CSCs that then cause a relapse of tumors [10]. Recently, it has been firmly established that CSCs are linked to epithelialmesenchymal transition (EMT), metastasis, drug resistance, progression and relapse of lung cancer [1115]. As a result, exploitation of the specific therapies targeting at CSCs has been a crucial issue in chemotherapeutic treatment of lung cancer. MicroRNAs (miRNAs) silence gene expression by binding to the 39-untranslated region of the target genes and have been reported to regulate CSCs self-renewal, tumorigenicity, metastasis, and chemoresistance in many human cancers [2,7,16]. For example, miR-34a repression causes colon CSCs to perform asymmetric cell division and promotes daughter cells to remain colon CSCs by regulating Notch signaling. Upregulated miR-143 in CSCs differentiation promotes prostate cancer cells metastasis by modulating FNDC3B expression. MiR-21 regulates EMT phenotype and hypoxia-inducible factor-1a expression in third-sphere forming breast cancer stem cell-like cells. MiR-200b, an important member of miR-200 families, is located at miR-200b/c/429 gene cluster, acts as a tumor suppressor in a variety of human solid tumors and has the capability of inhibiting CSCs growth and reversing the EMT phenotype of CSCs [17,18]. Recently, we have identified miR-200b as a key regulator of chemoresistance and restoration of miR-200b significantly reverses chemoresistance of docetaxel (DTX)-resistant LAD cells by inducing cell cycle arrest and apoptosis enhancement [19]. However, whether miR-200b regulates CSCs derived from docetaxel-resistant LAD cells is still poorly understood and needs to be further elucidated. In this study, we first show that miR-200b functions as a tumor suppressor both in vitro and in vivo in CSCs that are originated from human docetaxel-resistant LAD cells. Also, we identify HDAC1 as a specific regulator involved in silencing of miR-200b through a Sp1-dependent mechanism, and restoration of miR200b mediated by HDAC1 repression significantly suppresses maintenance of CSCs and reverses chemoresistance of CSCs by regulating Suz-12-E-cadherin signaling. To the best of our knowledge, there have been no reports about HDAC1/miR200b/Suz-12/E-cadherin regulatory network in regulating CSCs maintenance and chemoresistance in human LAD cells and the current work will provide a novel strategy for reversing chemoresistance of human LAD. Materials and Methods Ethics statement This study was approved by the research ethics committee of Jinling Hospital of NanJing University (Permit Number: 12-027) and was performed in compliance with the Helsinki Declaration. All animals were housed under specific pathogen-free conditions. All experimental procedures were performed in accordance with the Jinling Hospital of Nanjing University Guide for the Care and Use of Laboratory Animals. MicroBeads sorting Briefly, 3.06107 single cells were passed throug (...truncated)