DNA methylation transcriptionally regulates the putative tumor cell growth suppressor ZNF677 in non-small cell lung cancers

In our study, we investigated the role of ZNF677 in non-small cell lung cancers (NSCLC). By comparing ZNF677 expression in primary tumor (TU) and in the majority of cases also of corresponding non-malignant lung tissue (NL) samples from > 1,000 NSCLC patients, we found tumor-specific downregulation of ZNF677 expression (adjusted p-values < 0.001). We identified methylation as main mechanism for ZNF677 downregulation in NSCLC cells and we observed tumor-specific ZNF677 methylation in NSCLC patients (p < 0.0001). In the majority of TUs, ZNF677 methylation was associated with loss of ZNF677 expression. Moreover, ZNF677 overexpression in NSCLC cells was associated with reduced cell proliferation and cell migration. ZNF677 was identified to regulate expression of many genes mainly involved in growth hormone regulation and interferon signalling. Finally, patients with ZNF677 methylated TUs had a shorter overall survival compared to patients with ZNF677 not methylated TUs (p = 0.013). Overall, our results demonstrate that ZNF677 is trancriptionally regulated by methylation in NSCLCs, suggest that ZNF677 has tumor cell growth suppressing properties in NSCLCs and that ZNF677 methylation might serve as prognostic parameter in these patients

Identification and characterisation of epigenetically regulated putative tumor suppressor genes in non-small cell lung cancer

Weltweit sterben jährlich mehr als eineinhalb Millionen Menschen an Lungenkrebs, somit stellt Lungenkrebs die Haupttodesursache unter Krebserkrankungen dar. Dank der Entwicklung neuer Behandlungsmöglichkeiten und besserer diagnostischen Techniken, konnten die Überlebensraten von bestimmten Lungenkrebspatienten in den letzten Jahrzehnten verbessert werden. Viele der Patienten werden jedoch erst in einem fortgeschrittenen Stadium diagnostiziert, wo die Therapiemöglichkeiten begrenzt sind. Dementsprechend gibt es immer noch einen großen Bedarf neue Therapieansätze zu entwickeln, wobei die Voraussetzung dafür möglichst genaue Kenntnisse über die Mechanismen der Entstehung von Lungenkrebs sind. Ein Meilenstein in der modernen Krebsforschung war die Entdeckung der Epigenetik. Man erkannte, dass bei der Entstehung von Lungenkrebs nicht nur genetische Anomalien, sondern auch epigenetische Veränderungen eine wichtige Rolle spielen. Seitdem haben viele Studien gezeigt, dass vor allem fehlerhafte DNA Methylierung in der Promoterregion von wichtigen Tumorsuppressorgenen zu deren Funktionsverlust führt und somit zur Entstehung von nicht-kleinzelligem Lungenkarzinom beiträgt. Um die Rolle von fehlerhafter DNA Methylierung genauer zu untersuchen, haben wir die Methylierungsmuster von Primärtumoren und korrespondierendem normalen Lungengewebe von Lungenkrebspatienten auf genomweiter Ebene untersucht und verglichen. Mittels einer Technik, die Immunpräzipitation methylierter DNA und Microarray Analyse verbindet, konnten wir 477 tumor-spezifisch methylierte Gene in Patienten mit nicht-kleinzelligem Lungenkarzinom identifizieren. Viele dieser Gene spielen eine wichtige Rolle in unterschiedlichen biologischen Prozessen, deren Deregulation zur Krebsentstehung beitragen kann. Zusätzlich konnten wir zeigen, dass die Methylierung einiger der untersuchten Gene eine prognostische Relevanz für diese Patienten hat. In zwei darauffolgenden Projekten haben wir Gene selektioniert, deren tumor-spezifische Methylierung bislang unbekannt war und haben deren Methylierung in der Promoterregion, deren Protein- und Genexpression sowie deren Rolle in der Pathogenese vom nicht-kleinzelligem Lungenkarzinom genauer untersucht. Wir konnten zeigen, dass die Expression der Gene ZNF677, SPAG6 und L1TD1 hauptsächlich durch DNA Methylierung reguliert wird und dass ZNF677 und L1TD1 als potentielle Tumorsuppressorgene fungieren. Unsere Resultate zeigen, dass fehlerhafte DNA Methylierung für den Funktionsverlust einer Vielzahl von Genen verantwortlich ist und eine wichtige Rolle in der Pathogenese des nicht-kleinzelligen Lungenkarzinoms spielt.With more than 1.5 million deaths each year worldwide, lung cancer is still the leading cause of cancer-related deaths. Thanks to improved treatment strategies and newly developed diagnostic tools survival rates for certain lung cancer patients have improved over the last decades. However, the majority of lung cancer patients is diagnosed at advanced stages of the disease where treatment options are limited resulting in poor survival rates. Thus, there is a great need to identify molecular changes underlying lung cancer pathogenesis and to reveal potential new drug targets. A major step in lung cancer research was the identification of DNA methylation and how epigenetic modifications work in concert to regulate transcriptional activity. Since aberrant DNA methylation and histone modification patterns were frequently observed in genomes of cancer cells, cancer has been recognized not only as a genetic, but also as an epigenetic disease. Several studies demonstrated that aberrant DNA methylation is a frequently occurring event in non-small cell lung cancer and that crucial tumor suppressor genes are silenced by DNA methylation of their promoter regions. To gain more knowledge about how DNA methylation is involved in the pathogenesis of lung cancer we performed a genome-wide search for methylated CpG islands in primary tumors and corresponding normal lung tissue samples of non-small cell lung cancer patients. By the use of a technique that combines immunoprecipitation of methylated DNA with microarray analyses we identified 477 tumor-specifically methylated genes. Many of these genes were found to be involved in crucial biological processes including transcriptional control and cell adhesion. Furthermore, methylation of some of these genes may be of prognostic relevance for non-small cell lung cancer patients. In subsequent studies, we selected genes which were previously unknown to be methylated in non-small cell lung cancer and investigated their promoter methylation, gene and protein expression as well as their role in disease pathogenesis in detail. We identified DNA methylation as a main mechanism regulating transcriptional activity of ZNF677, SPAG6 and L1TD1 and showed that ZNF677 and L1TD1 may function as putative tumor suppressor genes in non-small cell lung cancer. Overall, our findings demonstrate the impact of DNA methylation in the pathogenesis of non-small cell lung cancer.submitted by Mag. Corinna AltenbergerZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2017OeB

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 IIII 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

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 © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Additional file 3: Figure S2. of SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

Impact of SPAG6 and L1TD1 mRNA expression on OS of NSCLC patients. (A) A shorter OS of squamous cell carcinoma patients with low SPAG6 mRNA expression (N = 155) compared to high SPAG6 mRNA expression (N = 267) was observed. (B) Adenocarcinoma patients with low L1TD1 mRNA expression (N = 138) showed a shorter OS compared to adenocarcinoma patients with high L1TD1 mRNA expression (N = 350). Gene expression microarray datasets (Affymetrix IDs 210032_s_at and 219955_at) were analysed and Kaplan-Meier plots were generated using all datasets and default settings of KM plotter. The cut-off values for “low” and “high” SPAG6 and L1TD1 mRNA expression were automatically defined by KM plotter software (Version 2013). (TIF 50 kb

Additional file 1: Table S1. of SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

Description of NSCLC cell lines used in this study. Information about histology, origin and disease stage of donors was obtained from ATCC catalogue ( https://www.lgcstandards-atcc.org ). EGFR, KRAS and TP53 mutational status and MET amplification according to supplementary references (1–3). *activating EGFR mutation in exon 19 (E746-E749 del), **activating EGFR mutation in exon 21 (L858R). N/A, not available; wt, wildtype; mut, mutated. Table S2. Clinico-pathological characteristics of 983 NSCLC patients. Overview of gender, histology, stage of disease and ethnicity of NSCLC patients obtained from TCGA database and used for mutation and copy number changes analyses of SPAG6 and L1TD1 is shown. ADC, adenocarcinoma; SCC, squamous cell carcinoma. Clinical data based on Caleydo software version 16/04/14. Table S3. Primer sequences. Summary of oligonucleotide sequences used for mRNA expression, MS-HRM, BGS analyses and construction of pCMV6-GFP expression vector. Y, random integration of C or T in fwd primer; R, random integration of G or A in rev primer. Table S4. Methylation of SPAG6 and L1TD1 in tumor cells of other tumor types. *Morphology, histology and origin of cell lines according to ATCC catalogue ( https://www.lgcstandards-atcc.org ). Percentage of methylation was calculated as described previously (4). (DOCX 33 kb

Additional file 2: Figure S1. of SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

SPAG6 and L1TD1 mRNA expression in different datasets of TCGA database. SPAG6 and L1TD1 mRNA expression was analysed using IlluminaHiSeq RNAseq data from TCGA database. Datasets LUAD and LUSC (lung), BRCA (breast), COADREAD (colorectal), HNSC (head and neck), KIRC (kidney), LIHC (liver) and PRAD (prostate) were analysed. Normalized log2 mRNA expression values are shown. Each dot represents a single sample. (TIF 176 kb

Additional file 4: Figure S3. of SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

SPAG6 and L1TD1 SNVs and deletions in NSCLC patients. TCGA LUAD and LUSC datasets were analysed with Caleydo software (version April 2014). Mutation of TP53 was used to demonstrate reliability of TCGA data analysis. ADC, adenocarcinoma patients; SCC, squamous carcinoma patients. (TIF 90 kb