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Supplementary Figures S1-S7. Effect of miR-634 overexpression in normal fibroblasts and LCLs (S1); Effect of NRF2 inhibition by specific siRNA transfection (S2); Simultaneous down-regulation of target genes by 7 siRNAs (S3); Expression analysis of miR-634 and target proteins in normal fibroblasts and LCLs (S4); Establishment of CDDP-resistant ESCC cell line and expression analysis of miR-634 and target proteins (S5); Changes in body weights of 6 mice in PBS group (S6); Expression analysis of miR-634 in ESCC samples (S7).

Supplementary Table S3. List of anti-apoptosis-, mitochondria-, and lysosome-related genes among the predicted target genes of miR-634.

Supplementary Table S1. List of the 626 predicted target genes of miR-634.

Supplementary Table S2. List of GO terms enriched among the predicted target genes of miR-634.

NRF2 is a master transcriptional regulator that integrates cellular stress responses and is negatively regulated by 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. Some tumor-suppressing microRNAs (miRNAs) target multiple oncogenes concurrently and therefore may be useful as cancer therapeutic agents. Further, such miRNAs may be useful to address chemotherapeutic resistance in cancer, which remains a primary clinical challenge in need of solutions. Thus, cytoprotective processes upregulated in cancer cells that are resistant to chemotherapy are a logical target for investigation. By using a reporter-coupled miRNA library screening, we have recently identified four miRNAs (miR-507, -634, -450a, and -129-5p) that negatively regulate the NRF2 activity by directly targeting. Importantly, downregulation 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). Interestingly, we found that overexpression of miR-634 activates the mitochondrial apoptotic pathway by direct concurrent targeting of genes associated with mitochondrial homeostasis, antiapoptosis, and autophagy, together with NRF2-mediated antioxidant ability. In particular, we showed how enforced expression of miR-634 enhanced chemotherapy-induced cytotoxicity in a model of ESCC, where resistance to chemotherapy remains clinically problematic. Our findings illustrate how reversing miRNA-mediated cytoprotective processes may offer a broadly useful approach to improving cancer therapy. Citation Format: Jun Inoue, Naoto Fujiwara, Shinsuke Yamamoto, Tatsuyuki Kawano, Johji Inazawa. MicroRNA-based diagnosis and therapy in NRF2-stabilized tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1064.

Lymph node metastasis (LNM) of esophageal squamous cell carcinoma (ESCC) is well-known to be an early event that is associated with poor prognosis of ESCC patients. In the last few years, tumor-specific aberrations in DNA methylation of CpG islands around promoter regions of tumor-related genes have been investigated as possible biomarkers for early diagnosis, treatment and prognosis of human cancers. But, there are few DNA methylation markers that can predict the presence of LNM in patients with ESCC. To identify DNA methylation markers correlated with LNM of ESCC, we performed a genome-wide screening of DNA methylation status in a discovery cohort of 67 primary ESCC tissues and their paired normal esophageal tissues by Illumina Infinium HumanMethylation450 BeadChip (covers more than 450,000 CpG sites). In this screening, we focused on some differentially methylated regions (DMRs), which were significantly associated with LNM of ESCC, as prime candidates for DNA methylation markers. In addition, these significant relationships were also confirmed by quantitative bisulfite pyrosequencing analysis in the same discovery cohort. Consequently, these results led us to consider that our selected DMRs might be useful epigenetic biomarkers for the prediction of the presence of LNM in primary ESCC cases. Therefore, we are continuing further analyses of these DMRs in an independent external validation cohort of ESCC patients. Citation Format: Ken-ichi Kozaki, Hiroaki Nagata, Kousuke Tanimoto, Naoto Fujiwara, Seiya Imoto, Daisuke Ichikawa, Satoru Miyano, Tatsuyuki Kawano, Eigo Otsuji, Johji Inazawa. Genome-wide screening of DNA methylation markers to predict the presence of lymph node metastasis in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1368. doi:10.1158/1538-7445.AM2014-1368

MicroRNAs (miRNAs) are endogenous small non-coding RNAs composed of 20∼25 base pairs that can negatively regulate gene expression by interfering with the translation of target transcripts. Since it has been known that some miRNAs function as tumor suppressors (TS) and their expression levels are often down-regulated in cancer, TS-miRNAs are expected to be therapeutic agents for cancer therapy. Thus, we conducted function-based screening using a miRNA library, and identified several TS-miRNAs, including miR-152, miR-218, miR-596. Recently, we identified miR-634 as a new TS-miRNA, which induced cell death with mitochondrial dysfunction and caspase activation. The expression analysis and the target-prediction program revealed miR-634 might directly target genes related with anti-apoptosis and mitochondrial function. Importantly, this miRNA was frequently down-regulated in primary tumor samples of esophageal squamous cell carcinoma (ESCC), compared with that in corresponding normal tissues. Furthermore, the combination therapy of miR-634 and cisplatin was effective to ESCC cell line in vitro and in vivo. Thus, these findings suggest that miR-634 may be useful as a potential therapeutic agent of ESCC. Citation Format: Naoto Fujiwara, Jun Inoue, Tatsuyuki Kawano, Ken-ichi Kozaki, Johji Inazawa. MicroRNA-634 induces caspase-dependent apoptosis by targeting anti-apopotic and mitochondria-related genes in human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5200. doi:10.1158/1538-7445.AM2014-5200