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PDF file - 148K, Expression analysis of 4 miRNAs in ESCC samples.

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, 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). 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. ©2013 AACR.

PDF file - 86K, Expression analysis for NRF2 expression in miRNA-transfected cells.

PDF file - 115K, Identification of transcriptional target genes of NRF2 as the target of miR-507.

XLSX file - 94K, Result of function-based screening of miRNAs negatively regulating transcriptional activity of NRF2 in HeLa cells used in Pre-miR? miRNA Precursor Library - Human V4 (Ambion).

PDF file - 113K, Expression analysis of NRF2 target genes.

PDF file - 94K, Expression analysis of the four miRNAs and the effect of transfection of miR-507 on cell survival under cellular stress caused by Cisplatin (CDDP) treatment (left) in A549 cells.

XLSX file - 34K, Primers for qRT-PCR and mutation analysis.

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XLSX file - 29K, Primers for human NQO1 promoter region.

Purpose: The aim of this study was the identification of novel tumor suppressor genes (TSG) silenced by DNA hypermethylation in hepatocellular carcinoma (HCC).Experimental Design: We conducted integrative array-based approach for genome-wide screening of methylation targets using a methylated DNA immunoprecipitation-CpG island microarray and expression array in three universal hepatoma cell lines and normal liver tissue. Through detailed expression and functional analyses using hepatoma cell lines and primary HCC samples, we isolated novel TSGs for HCC.Results: A total of 642 genes were identified as methylated in three hepatoma cell lines but unmethylated in normal liver tissue, whereas 204 genes on autosomes were identified as genes unexpressed but restored after treatment with 5-aza-2′-deoxycytidine in these cell lines and expressed in normal tissue. Through the integration of results of the two-array analyses and further validation analyses of expression and methylation status in 17 cell lines and 30 primary tumors of hepatoma, we identified MZB1, marginal zone B and B1 cell-specific protein, encoding an endoplasmic reticulum protein, as a putative TSG frequently methylated within its CpG island in hepatoma. Among 162 patients with primary HCC, silencing of MZB1 protein was significantly and independently associated with a worse outcome. Restoration of MZB1 expression in hepatoma cells reduced cell proliferation in vitro and in vivo through G1-arrest.Conclusions: These results suggest that methylation-mediated silencing of MZB1 expression leads to loss of its tumor-suppressive activity, which may be a factor in the hepatocarcinogenesis, and is a useful prognosticator in HCC. Clin Cancer Res; 18(13); 3541–51. ©2012 AACR.

PDF file, 37KB, Primers for COBRA and bisulfite sequencing(bisulfite-PCR).

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PDF file, 55KB, Positive (methylated) and negative (unmethylated) control gene set for MeDIP-chip analysis.

PDF file, 245KB, S1 Determination of the cut-off value to accurately distinguish methylated genes in MeDIP-chip analyses.; S2 Profiles of relative expression of 11 candidates determined by quantitative real-time RT-PCR in three hepatoma cell lines (Hep3B, HepG2, and HuH-7) treated with 1, 5, and 10 μM of 5-aza-dCyd.; S3 Representative results of the methylation status of Region 3 of FAR1 CpG-island determined by COBRA (upper) and the relative expression level of FAR1 mRNA determined by quantitative real-time RT-PCR (lower) in surgically resected primary HCC tumors (T) and corresponding non-cancerous liver tissues (N).; S4 Representative results of BGS of the MZB1 CpG island (Region 1-8) in HLE cells without and with 10 μM 5-aza-dCyd.

PDF file, 60KB, Eleven candidates for epigenetically silenced genes in HCC cells identified through our integrated array-based approach.

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Supplementary Table 2 from Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

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

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Supplementary Table 1 from Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

Supplementary Table 4 from Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

Supplementary Figure 1 from Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

Supplementary Table 1 from Exploration of Tumor-Suppressive MicroRNAs Silenced by DNA Hypermethylation in Oral Cancer

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The etiology and development of human cancers that remain little understood might be enlightened by defining tumor suppressor microRNAs (TS-miRNA). In this study, we identified TS-miRNAs silenced by aberrant DNA hypermethylation in endometrial cancer. Functional screening of 327 synthetic miRNAs in an endometrial cancer cell proliferation assay identified 103 miRNAs that inhibited cell growth. We then determined the sequence, DNA methylation status, and expression levels of these miRNAs in endometrial cancer cell lines and primary tumors. These determinations led to the identification of miR-152 as a candidate TS-miRNA gene in endometrial cancer. Epigenetic silencing documented in miR-152 was consistent with its location at 17q21.32 in intron 1 of the COPZ2 gene, which is also silenced often in endometrial cancer by DNA hypermethylation, and also with evidence that miR-152 targets the DNA methyltransferase DNMT1. Notably, restoration of miR-152 expression in endometrial cancer cell lines was sufficient to inhibit tumor cell growth in vitro and in vivo. We identified E2F3, MET, and Rictor as novel candidate targets of miR-152, suggesting how its epigenetic silencing can drive endometrial carcinogenesis. Our findings define a central role for miR-152 in endometrial cancer, and they also suggest its use in new therapeutic strategies to treat this cancer. Cancer Res; 71(20); 6450–62. ©2011 AACR.

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Angiopoietin-like protein 2 (ANGPTL2) is a secreted protein belonging to the angiopoietin family, the members of which are implicated in various biological processes, although its receptor remains unknown. We identified a homozygous loss of ANGPTL2 (9q33.3) in the course of screening a panel of ovarian cancer (OC) cell lines for genomic copy-number aberrations using in-house array-based comparative genomic hybridization. ANGPTL2 mRNA expression was observed in normal ovarian tissue and immortalized normal ovarian epithelial cells, but was reduced in some OC lines without its homozygous deletion (18 of 23 lines) and restored after treatment with 5-aza 2′-deoxycytidine. The methylation status of sequences around the ANGPTL2 CpG-island with clear promoter activity inversely correlated with expression. ANGPTL2 methylation was frequently observed in primary OC tissues as well. In an immunohistochemical analysis of primary OCs, ANGPTL2 expression was frequently reduced (51 of 100 cases), and inversely correlated with methylation status. Patients with OC showing reduced ANGPTL2 immunoreactivity had significantly worse survival in the earlier stages (stages I and II), but better survival in advanced stages (stages III and IV). The restoration of ANGPTL2 expression or treatment with conditioned medium containing ANGPTL2 inhibited the growth of OC cells originally lacking the expression of this gene, whereas the knockdown of endogenous ANGPTL2 accelerated the growth of OC cells with the expression of ANGPTL2. These results suggest that, at least partly, epigenetic silencing by hypermethylation of the ANGPTL2 promoter leads to a loss of ANGPTL2 function, which may be a factor in the carcinogenesis of OC in a stage-dependent manner. [Cancer Res 2008;68(13):5067–75]

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Supplementary Figure 4 from Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

Supplementary Figure 2 from Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

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In the last few years, microRNAs (miRNA) have started a revolution in molecular biology and emerged as key players in the carcinogenesis. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. To identify the miRNA signature that was specific for oral squamous cell carcinoma (OSCC), we first examined expression profiles of 148 miRNAs in a panel of 18 OSCC cell lines and the immortalized oral keratinocyte line RT7 as a control. Compared with RT7, the expression of 54 miRNAs (36.5%) was frequently down-regulated in OSCC lines (miR-34b, miR-137, miR-193a, and miR-203), located around CpG islands, to identify tumor-suppressive miRNAs silenced through aberrant DNA methylation. The expression of those four genes was restored by treatment with 5-aza-2′-deoxycytidine in OSCC cells lacking their expression. In addition, expression levels of the four miRNAs were inversely correlated with their DNA methylation status in the OSCC lines. In primary tumors of OSCC with paired normal oral mucosa, down-regulation of miRNA expression through tumor-specific hypermethylation was more frequently observed for miR-137 and miR-193a than for miR-34b and miR-203. Moreover, the ectopic transfection of miR-137 or miR-193a into OSCC lines lacking their expressions significantly reduced cell growth, with down-regulation of the translation of cyclin-dependent kinase 6 or E2F transcription factor 6, respectively. Taken together, our results clearly show that miR-137 and miR-193a are tumor suppressor miRNAs epigenetically silenced during oral carcinogenesis. [Cancer Res 2008;68(7):2094–105]

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Supplementary Table 3 from Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

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Supplementary Table 5 from Exploration of Tumor-Suppressive MicroRNAs Silenced by DNA Hypermethylation in Oral Cancer

Supplementary Table 2 from Exploration of Tumor-Suppressive MicroRNAs Silenced by DNA Hypermethylation in Oral Cancer

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).

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Supplementary Table 4 from Exploration of Tumor-Suppressive MicroRNAs Silenced by DNA Hypermethylation in Oral Cancer

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Supplementary Figure 3 from Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

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