Cell Death and Survival The Impact of miRNA-Based Molecular Diagnostics and Treatment of NRF2-Stabilized Tumors Shinsuke Yamamoto 1,6 , Jun Inoue 1 , Tatsuyuki Kawano 5 , Ken-ichi Kozaki 1,2 , Ken Omura 3,4,6 , and Johji Inazawa 1,2,4 Abstract NF-E2–related factor 2 (NRF2) is a master transcriptional regulator that integrates cellular stress responses and is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1) at the posttranslational level. In human cancers, aberrantly stabilized NRF2, either by mutation of NRF2 or KEAP1, plays a vital role in chemoresistance and tumor cell growth through the transcriptional activation of target genes, suggesting that targeted inhibition of NRF2 is a potential therapy for NRF2-stabilized tumors. MicroRNAs (miRNA) are endogenous small noncoding RNAs that can negatively regulate gene expression by interfering with the translation or stability of target transcripts. Moreover, tumor-suppressor miRNAs have been suggested to be useful for cancer treatment. Here, a reporter-coupled miRNA library screen identified four miRNAs (miR-507, -634, -450a, and -129-5p) that negatively regulate the NRF2-mediated oncogenic pathway by directly targeting NRF2. Importantly, down- regulation of these miRNAs, in addition to the somatic mutation of NRF2 or KEAP1, is associated with stabilized NRF2 and poor prognosis in esophageal squamous cell carcinoma (ESCC). Furthermore, administration of a miR-507 alone or in combination with cisplatin inhibited tumor growth in vivo. Thus, these findings reveal that miRNA-based therapy is effective against NRF2-stabilized ESCC tumors. Implications: This study determines the potential of miRNA-based molecular diagnostics and therapeutics in NRF2-stablized tumors. Mol Cancer Res; 12(1); 58–68. Ó2013 AACR. Introduction NF-E2–related factor 2 (NRF2) is a master transcriptional regulator for cytoprotection against cellular damage from chemotherapy and oxidative stress (1, 2). Under physiologic conditions, NRF2 is ubiquitinated by the cullin 3 (CUL3) Kelch-like ECH-associated protein 1 (KEAP1) ubiquitin E3 ligase complex and is constantly degraded in the proteasome, resulting in a low cellular concentration of the NRF2 protein. Under cellular stress, KEAP1 is inactivated and NRF2 is stabilized in the nucleus, resulting in cell survival through the transcriptional activation of target genes to which NRF2 binds directly to the antioxidative responsive element (ARE) within the promoter in each of the target genes (3, 4). In addition to cellular stress response, it has been recently reported that NRF2 can also contribute to tumor cell growth by modulating metabolism (5). It has been found that gene mutations leading to a gain-of-function for NRF2 or loss-of-function for KEAP1 in various types of human cancers result in NRF2-mediated cancer cell survival and growth due to the constitutive activation of NRF2 (6–9). Interestingly, excess accumulation (as the aggregate) of the p62 protein, a substrate for protein degradation by autop- hagy, may also stabilize NRF2 by competitively interacting with KEAP1 (10–13). Thus, NRF2 has an oncogenic function in cancer cells, and a high level of NRF2 protein is associated with a poor prognosis (14–16). On the basis of this evidence, the therapeutic inhibition of the NRF2- mediated oncogenic pathway may benefit patients with NRF2-stabilized tumors. MicroRNAs (miRNA) are endogenous small noncoding RNAs that regulate gene expression by interfering with the translation or stability of target transcripts through bind- ing to the 3 0 -untranslated region (UTR; refs. 17, 18). Some miRNAs can negatively regulate oncogene(s), and the downregulation of tumor-suppressive miRNAs leads to the activation of oncogenic pathways (19–22). Indeed, we have identified novel tumor-suppressive miRNAs in endometrial cancer and oral squamous cell carcinoma through function-based screening using double-stranded RNAs (dsRNA) mimicking sequences of mature miRNAs Authors' Affiliations: 1 Department of Molecular Cytogenetics, Medical Research Institute and Graduate School of Medical and Dental Sciences; Departments of 2 Genome Medicine and 3 Advanced Molecular Diagnosis and Maxillofacial Surgery, Hard Tissue Genome Research Center; 4 Global Center of Excellence Program for International Research Center for Molec- ular Science in Tooth and Bone Diseases, Departments of 5 Surgery and 6 Oral and Maxillofacial Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Johji Inazawa, Department of Molecular Cytoge- netics, Medical Research Institute, Tokyo Medical and Dental University, 1- 5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 81-358-035- 820; Fax: 81-358-030-244; E-mail: johinaz.cgen@mri.tmd.ac.jp doi: 10.1158/1541-7786.MCR-13-0246-T Ó2013 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 12(1) January 2014 58 Downloaded from http://aacrjournals.org/mcr/article-pdf/12/1/58/3135812/58.pdf by guest on 18 November 2023