Barbara Ziegler

Genome-Wide miRNA Expression Profiling Identifies miR-9-3 and miR-193a as Targets for DNA Methylation in Non–Small Cell Lung Cancers

Purpose: The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on miRNA silencing in non–small cell lung cancers (NSCLC). Experimental Design: We conducted microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. miRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding nonmalignant lung tissue samples of 101 patients with stage I–III NSCLC. Results: By comparing microarray data of untreated and drug-treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analyzed. Moreover, miR-9-3 and miR-193a were found to be tumor specifically methylated in patients with NSCLC. We observed a shorter disease-free survival of patients with miR-9-3 methylated lung squamous cell carcinoma (LSCC) than patients with miR-9-3 unmethylated LSCC by multivariate analysis [HR = 3.8; 95% confidence interval (CI), 1.3–11.2, P = 0.017] and a shorter overall survival of patients with miR-9-3 methylated LSCC than patients with miR-9-3 unmethylated LSCC by univariate analysis (P = 0.013). Conclusions: Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in patients with LSCC. Clin Cancer Res; 18(6); 1619–29. ©2012 AACR.

Genome-wide CpG island methylation analyses in non-small cell lung cancer patients

DNA methylation is part of the epigenetic gene regulation complex, which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stages I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis. Overall, we identified 2414 genomic positions differentially methylated between tumor and non-malignant lung tissue samples. Ninety-seven percent of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes of which many are involved in regulation of gene transcription and cell adhesion. Tumor-specific methylation was confirmed by a gene-specific approach. In the majority of tumors, methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analyzed by gene expression microarrays suggesting that about one-third of these genes are transcriptionally regulated by methylation. Moreover, comparison of methylation results with certain clinicopathological characteristics of the patients suggests that methylation of HOXA2 and HOXA10 may be of prognostic relevance in squamous cell carcinoma (SCC) patients. In conclusion, we identified a large number of tumor-specifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Moreover, HOXA2 and HOXA10 methylation may serve as prognostic parameters in SCC patients. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs.

Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic myeloid leukemia

Little is known about the impact of DNA methylation on the evolution/progression of Ph+ chronic myeloid leukemia (CML). We investigated the methylome of CML patients in chronic phase (CP-CML), accelerated phase (AP-CML) and blast crisis (BC-CML) as well as in controls by reduced representation bisulfite sequencing. Although only ~600 differentially methylated CpG sites were identified in samples obtained from CP-CML patients compared with controls, ~6500 differentially methylated CpG sites were found in samples from BC-CML patients. In the majority of affected CpG sites, methylation was increased. In CP-CML patients who progressed to AP-CML/BC-CML, we identified up to 897 genes that were methylated at the time of progression but not at the time of diagnosis. Using RNA-sequencing, we observed downregulated expression of many of these genes in BC-CML compared with CP-CML samples. Several of them are well-known tumor-suppressor genes or regulators of cell proliferation, and gene re-expression was observed by the use of epigenetic active drugs. Together, our results demonstrate that CpG site methylation clearly increases during CML progression and that it may provide a useful basis for revealing new targets of therapy in advanced CML.

Epigenetic down-regulation of integrin α7 increases migratory potential and confers poor prognosis in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a devastating malignancy characterized by invasive growth and rapid recurrence. The identification and inhibition of molecular components leading to this migratory and invasive phenotype are thus essential. Accordingly, a genome‐wide expression array analysis was performed on MPM cell lines and a set of 139 genes was identified as differentially expressed in cells with high versus low migratory activity. Reduced expression of the novel tumour suppressor integrin α7 (ITGA7) was found in highly motile cells. A significant negative correlation was observed between ITGA7 transcript levels and average displacement of cells. Forced overexpression of ITGA7 in MPM cells with low endogenous ITGA7 expression inhibited cell motility, providing direct evidence for the regulatory role of ITGA7 in MPM cell migration. MPM cells showed decreased ITGA7 expressions at both transcription and protein levels when compared to non‐malignant mesothelial cells. The majority of MPM cell cultures displayed hypermethylation of the ITGA7 promoter when compared to mesothelial cultures. A statistically significant negative correlation between ITGA7 methylation and ITGA7 expression was also observed in MPM cells. While normal human pleura samples unambiguously expressed ITGA7, a varying level of expression was found in a panel of 200 human MPM samples. In multivariate analysis, ITGA7 expression was found to be an independent prognostic factor. Although there was no correlation between histological subtypes and ITGA7 expression, importantly, patients with high tumour cell ITGA7 expression had an increased median overall survival compared to the low‐ or no‐expression groups (463 versus 278 days). In conclusion, our data suggest that ITGA7 is an epigenetically regulated tumour suppressor gene and a prognostic factor in human MPM. Copyright

EVI1 promotes tumor growth via transcriptional repression of MS4A3.

BackgroundThe transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells.MethodsU937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice.ResultsGene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis.ConclusionsOur data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.

Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples.

DNA methylation of microRNA-coding genes in non-small-cell lung cancer patients: MicroRNA methylation in NSCLC patients

Deregulated DNA methylation leading to transcriptional inactivation of certain genes occurs frequently in non‐small‐cell lung cancers (NSCLCs). As well as protein‐coding genes, microRNA (miRNA)‐coding genes may be targets for methylation in NSCLCs; however, the number of known methylated miRNA genes is still small. Thus, we investigated methylation of miRNA genes in primary tumour (TU) samples and corresponding non‐malignant lung tissue (NL) samples of 50 NSCLC patients by using methylated DNA immunoprecipitation followed by custom‐designed tiling microarray analyses (MeDIP‐chip), and 252 differentially methylated probes between TU samples and NL samples were identified. These probes were annotated, which resulted in the identification of 34 miRNA genes with increased methylation in TU samples. Some of these miRNA genes were already known to be methylated in NSCLCs (e.g. those encoding miR‐9‐3 and miR‐124), but methylation of the vast majority of them was previously unknown. We selected six miRNA genes (those encoding miR‐10b, miR‐1179, miR‐137, miR‐572, miR‐3150b, and miR‐129‐2) for gene‐specific methylation analyses in TU samples and corresponding NL samples of 104 NSCLC patients, and observed a statistically significant increase in methylation of these genes in TU samples (p < 0.0001). In silico target prediction of the six miRNAs identified several oncogenic/cell proliferation‐promoting factors (e.g. CCNE1 as an miR‐1179 target). To investigate whether miR‐1179 indeed targets CCNE1, we transfected miR‐1179 gene mimics into CCNE1‐expressing NSCLC cells, and observed downregulated CCNE1 mRNA expression in these cells as compared with control cells. Similar effects on cyclin E1 expression were seen in western blot analyses. In addition, we found a statistically significant reduction in the growth of NSCLC cells transfected with miR‐1179 mimics as compared with control cells. In conclusion, we identified many methylated miRNA genes in NSCLC patients, and found that the miR‐1179 gene is a potential tumour cell growth suppressor in NSCLCs. Overall, our findings emphasize the impact of miRNA gene methylation on the pathogenesis of NSCLCs.

Abstract 2766: Genome-wide miRNA methylation analyses in non-small cell lung cancer patients

Deregulated DNA methylation (referred to as methylation) leading to transcriptional inactivation of certain genes occurs frequently in non-small cell lung cancers (NSCLC). Besides protein encoding genes also microRNA (miRNA) encoding genes were found to be targets for methylation in NSCLCs, however, the number of known methylated miRNA genes is still small. Thus, we investigated genome-wide methylation of miRNA genes in primary tumors (TU) and corresponding non-malignant lung tissue samples (NL) of 50 NSCLC patients using methylated DNA immunoprecipitation followed by custom designed tiling microarray analyses (MeDIP-chip). Differentially methylated miRNA genes between TU and NL samples were identified using paired t-statistics with permutation adjusted p-values for step down multiple testing. Using this approach, 201 differentially methylated probes between TU and NL samples were found. These probes were annotated resulting in the identification of 39 tumor-specifically methylated miRNA genes. 79% of them are associated with a 5′ CpG island. While some of these miRNA genes were already known to be methylated in NSCLCs (e.g. miR-9-3, miR-124) methylation of the vast majority of them was unknown so far. We selected 6 miRNA genes (miR-10b, miR-1179, miR-137, miR-572, miR-3150b and miR-129-2) for gene-specific methylation analyses in TU and corresponding NL samples of 108 NSCLC patients and observed statistically significant tumor-specific methylation of these miRNA genes which confirmed our MeDIP-chip data (p 1.000 NSCLC patients from The Cancer Genome Atlas database, we found that many of the predicted miRNA targets are upregulated in TU samples (e.g. CCNE1, HMGB3, HOXD11, TFAP2A, ZIC2). Based on these findings, we investigated if miR-1179 indeed targets CCNE1 in vitro. Thus, we transfected miR-1179 mimics into CCNE1 expressing NSCLC cell lines (HTB182 and NCI-H1650). Using RT-PCR, we found downregulated CCNE1 expression in cells transfected with miR-1179 mimics compared to cells transfected with controls. In addition, we observed downregulated CCNE1 expression in cells transfected with miR-1179 mimics compared to cells transfected with controls by Western blot analyses. Additional functional analyses of miR-1179 targets as well as of other miRNA targets are ongoing. In conclusion, we identified a large number of tumor-specifically methylated miRNA genes in NSCLC patients and found that some of these miRNAs target certain oncogenes in NSCLC cells. Overall, our findings emphasize the impact of miRNA gene methylation on the pathogenesis of NSCLCs. Citation Format: Gerwin Heller, Corinna Altenberger, Thais Topakian, Barbara Ziegler, Gyorgy Lang, Adelheid End-Pfutzenreuter, Irene Steiner, Sonja Zehetmayer, Balazs Dome, Walter Klepetko, Martin Posch, Christoph C. Zielinski, Sabine Zochbauer-Muller. Genome-wide miRNA methylation analyses in non-small cell lung cancer patients. [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 2766.

Abstract 2772: SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

DNA methylation is one of the major epigenetic mechanisms regulating transcriptional activity and is involved in the pathogenesis of non-small cell lung cancers (NSCLC). In a recent study we identified a large number of tumor-specifically methylated genes in NSCLCs (Heller et al., Carcinogenesis 2013). For further detailed analyses we selected the genes SPAG6 (Sperm Associated Antigen 6) and L1TD1 (LINE-1 Type Transposase Domain Containing 1). By analysing publically available IlluminaHiSeq RNA-seq data we observed frequent downregulation of SPAG6 and L1TD1 mRNA expression in primary tumor (TU) samples compared to corresponding non-malignant lung tissue (NL) samples of NSCLC patients. In addition, we investigated SPAG6 and L1TD1 mRNA expression in 5 NSCLC cell lines and found SPAG6 as well as L1TD1 mRNA expression frequently downregulated in all of these cell lines compared to normal human bronchial epithelial cells (NHBECs). Subsequently, we treated cells of NSCLC cell lines which did not express SPAG6 or L1TD1 with the epigenetically active drugs 5-aza-2′-deoxycytidine and Trichostatin A and observed re-expression of both genes suggesting that transcriptional regulation of SPAG6 and L1TD1 is mediated by DNA methylation in NSCLCs. Bisulfite genomic sequencing of parts of the 5′ region of SPAG6 and L1TD1 revealed that the vast majority of CpG sites indeed are methylated in NSCLC cells in contrast to NHBECs. Moreover, we analysed SPAG6 and L1TD1 methylation in TU and NL samples of 147 stage I-III NSCLC patients using the gene-specific approach methylation-sensitive high resolution melt analysis (MS-HRM). Differences in SPAG6 as well as in L1TD1 methylation between TU and NL samples were statistically significant for both genes and confirmed that SPAG6 and L1TD1 are tumor-specifically methylated in NSCLCs. Additionally, we investigated SPAG6 and L1TD1 protein expression in TU and NL samples of 35 NSCLC patients by immunohistochemistry. In the vast majority of SPAG6 or L1TD1 methylated TU samples, protein expression of these genes was downregulated in tumor cells. Moreover, we performed cell proliferation, cell viability and colony formation assays in vitro and observed that ectopic expression of L1TD1 but not of SPAG6 reduced tumor cell proliferation, viability and the ability of NSCLC cells to form colonies. To further investigate the role of L1TD1 in the pathogenesis of NSCLCs, in vivo studies are being carried out. Overall, our results demonstrate that DNA methylation is the major mechanism for frequent downregulation of SPAG6 and L1TD1 expression in NSCLCs. Citation Format: Corinna Altenberger, Gerwin Heller, Barbara Ziegler, Erwin Tomasich, Maximilian Marhold, Leonhard Mullauer, Gyorgy Lang, Adelheid End-Pfutzenreuter, Balazs Dome, Britt-Madeleine Arns, Walter Klepetko, Christoph C. Zielinski, Sabine Zochbauer-Muller. SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers. [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 2772.

Abstract 397: Transcriptional regulation of SPAG6 by DNA methylation in NSCLCs

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Epigenetic abnormalities, especially DNA methylation, are involved in the pathogenesis of non-small cell lung cancers (NSCLC). By using a genome-wide approach, we recently identified ∼ 500 tumor-specifically methylated genes in a large number of NSCLC patients and from most of them tumor-specific methylation in NSCLCs was unknown so far. One of these genes is the Sperm Associated Antigen 6 (SPAG6) gene. By analysing publically available IlluminaHiSeq RNA-seq data from “The Cancer Genome Atlas” database, we observed that SPAG6 mRNA expression is frequently lost in tumor samples compared to corresponding non-malignant lung tissue samples of NSCLC patients (http://cancergenome.nih.gov/). To further investigate the mechanism of SPAG6 downregulation, we studied SPAG6 expression in 5 NSCLC cell lines. We observed loss of SPAG6 expression in all of these cell lines compared to normal human bronchial epithelial cells (NHBECs). Subsequently, we treated cells of NSCLC cell lines which do not express SPAG6 with the epigenetically active drugs 5-aza-2´-deoxycytidine and Trichostatin A and observed SPAG6 reexpression. These results suggest that transcriptional regulation of SPAG6 is epigenetically regulated. Bisulfite genomic sequencing of parts of the 5´ region of SPAG6 revealed that the vast majority of CpG sites indeed are methylated in cells of NSCLC cell lines which do not express SPAG6 while no methylation was found in NHBECs. Moroever, we analysed SPAG6 methylation in tumor and corresponding non-malignant tissue samples of 147 stage I-III NSCLC patients using methylation-sensitive high-resolution melting (MS-HRM) assays. Differences in SPAG6 methylation between tumor and corresponding non-malignant lung tissue samples were statistically significant (p < 0.0001) demonstrating that SPAG6 is tumor-specifically methylated. Furthermore, ROC curve analyses of methylation results revealed that methylation of SPAG6 is able to distinguish tumor samples from corresponding non-malignant lung tissue samples of NSCLC patients. We additionally investigated SPAG6 protein expression in tumor and corresponding non-malignant tissue samples of 35 NSCLC patients by immunohistochemistry. In the vast majority of tumor samples which were found to be SPAG6 methylated, SPAG6 protein expression was lost in tumor cells while SPAG6 protein expression was observed in bronchial and bronchiolar epithelial cells of non-malignant lung tissue samples. Comparison of SPAG6 methylation results with clinico-pathological data of NSCLC patients suggests a potential negative prognostic relevance of SPAG6 methylation for NSCLC patients. Currently, we determine the biological function of SPAG6 in NSCLC cell lines. Overall, our results demonstrate that DNA methylation is the major mechanism for frequent loss of SPAG6 expression in NSCLCs. Citation Format: Corinna Altenberger, Gerwin Heller, Bianca Schmid, Barbara Ziegler, Leonhard Mullauer, Gyorgy Lang, Adelheid End-Pfutzenreuter, Balazs Dome, Britt-Madeleine Arns, Kwun M. Fong, Casey M. Wright, Ian A. Yang, Walter Klepetko, Christoph C. Zielinski, Sabine Zochbauer-Muller. Transcriptional regulation of SPAG6 by DNA methylation in NSCLCs. [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 397. doi:10.1158/1538-7445.AM2014-397