Epigenetische Alterationen des Lungenkarzinoms: Diagnostische Nutzung und Induktion durch E-Zigarettenkonsum

Das nicht-kleinzellige Lungenkarzinom (engl. non-small cell lung cancer; NSCLC) ist die zweithäufigste Krebsart bei Männern und Frauen. NSCLC wird häufig im fortgeschrittenen Stadium diagnostiziert und ist mit einer schlechten Prognose assoziiert. Rauchen zählt zu den häufigsten Ursachen für die Entstehung von Bronchialkarzinomen, weswegen heutzutage viele Raucher zur E-Zigarette wechseln. Da beim Verdampfen des Liquids u.a. Formaldehyd und Acrolein entstehen, raten einige Studien vom E-Zigarettenkonsum ab. Die vorliegende Doktorarbeit wurde im Rahmen des Deutschen Zentrums für Lungenkrankheiten (DZL) durchgeführt. Ziel war es, einerseits einen zuverlässigen Früherkennungstest anhand von bestimmten, für das Lungenkarzinom spezifischen Epimutationen zu entwickeln. Anderseits wurde die Wirkung des E-Zigarettendampfes auf die Entstehung von Epimutationen untersucht. Bei dem entwickelten Test für Lungenkrebsfrüherkennung handelte es sich um ein auf Liquid Biopsies basiertes NGS-Panel, das 638 Regionen umfasste. Um das NGS-Panel zu entwickeln wurden DNA-Methylierungsmuster in primären Tumoren und Blutplasma analysiert. Im Rahmen der Pilot-Studie differenzierte das entwickelte Plasma Panel mit einer 100%igen Richtigkeit maligne Lungentumore (Stadium: I bis IV) und lieferte weitere Informationen bezüglich deren Entität und Staging. Die Ergebnisse dieser Arbeit stellen damit eine wesentliche Grundlage für die erfolgreiche Beantragung der deutschlandweiten Clinical Trial Studie „EmoLung“ des DZL dar. Um die Wirkung des E-Zigarettendampfes auf Lungenzellen zu untersuchen, wurde eine multi-OMICS Studie designt, in der primäre Lungenepithelzellen sowie die A549 Lungenzelllinie parallel analysiert wurden. Dabei konnte mit einer Kombination epigenetischer, molekularbiologischer und zellphysiologischer Untersuchungen erstmals eine Proliferations- und Aneuploidie fördernde Wirkung durch E-Zigarettenextrakt gezeigt werden.Non-small cell lung cancer (NSCLC) is worldwide the second most common cancer in both men and women. NSCLC is often diagnosed in advanced stages and is therefore associated with poor prognosis. Smoking is one of the most frequent causes of bronchial carcinomas. As a consequence many smokers switch to e-cigarettes. However, during vapouring big amounts of formaldehyde and acrolein develop. For this reason several studies disadvise e-cigarette consumption. This work was performed in the framework of The German Center for Lung Research (DZL). The aims were to design a reliable, epimutation-based test for lung cancer early detection and to analyse the effect of e-cigarette vapour on the appearance of those epimutations. The developed lung cancer early detection test was a liquid biopsy-based NGS-panel, which included 638 regions. During the development DNA methylation signatures in tumors and blood plasma were analysed. The developed panel identified in a pilot study lung cancer patients (stage I to IV) with an accuracy of 100%. Moreover, it provided information about the entity and the tumor stage. The results of this study provided the basis for a successful application of the German Clinical Trial DZL-Study „EmoLung“. To analyse the effect of e-cigarette vapour on primary epithelial lung cells as well as A549 cell line, a multi-OMICS study was designed. By using epigenetic, molecular and physiological approaches, the multi-OMICS study showed for the first time a proliferative and aneuploidy promoting effect of e-cigarettes

DNA methylation profiles of bronchoscopic biopsies for the diagnosis of lung cancer

Background!#!Lung cancer is the leading cause of cancer-related death in most western countries in both, males and females, accounting for roughly 20-25% of all cancer deaths. For choosing the most appropriate therapy regimen a definite diagnosis is a prerequisite. However, histological characterization of bronchoscopic biopsies particularly with low tumor cell content is often challenging. Therefore, this study aims at (a) determining the value of DNA methylation analysis applied to specimens obtained by bronchoscopic biopsy for the diagnosis of lung cancer and (b) at comparing aberrantly CpG loci identified in bronchoscopic biopsy with those identified by analyzing surgical specimens.!##!Results!#!We report the HumanMethylation450-based DNA methylation analysis of paired samples of bronchoscopic biopsy specimens either from the tumor side or from the contralateral tumor-free bronchus in 37 patients with definite lung cancer diagnosis and 18 patients with suspicious diagnosis. A differential DNA methylation analysis between both biopsy sites of patients with definite diagnosis identified 1303 loci. Even those samples were separated by the set of 1303 loci in which histopathological analysis could not unambiguously define the dignity. Further differential DNA methylation analyses distinguished between SCLC and NSCLC. We validated our results in an independent cohort of 40 primary lung cancers obtained by open surgical resection and their corresponding controls from the same patient as well as in publically available DNA methylation data from a TCGA cohort which could also be classified with high accuracy.!##!Conclusions!#!Considering that the prognosis correlates with tumor stage at time of diagnosis, early detection of lung cancer is vital and DNA methylation analysis might add valuable information to reliably characterize lung cancer even in histologically ambiguous sample material

Downregulation of the TGF-beta pseudoreceptor BAMBI in non-small cell lung cancer enhances TGF-beta signaling and invasion.

Non-small cell lung cancer (NSCLC) is characterized by early metastasis and has the highest mortality rate among all solid tumors, with the majority of patients diagnosed at an advanced stage where curative therapeutic options are lacking. In this study, we identify a targetable mechanism involving TGF-beta elevation that orchestrates tumor progression in this disease. Substantial activation of this pathway was detected in human lung cancer tissues with concomitant downregulation of BAMBI, a negative regulator of the TGF-beta signaling pathway. Alterations of epithelial-to-mesenchymal transition (EMT) marker expression were observed in lung cancer samples compared to tumor-free tissues. Distinct alterations in the DNA methylation of the gene regions encoding TGF-beta pathway components were detected in NSCLC samples compared to tumor-free lung tissues. In particular, epigenetic silencing of BAMBI was identified as a hallmark of NSCLC. Reconstitution of BAMBI expression in NSCLC cells resulted in a marked reduction of TGF-beta-induced EMT, migration and invasion in vitro, along with reduced tumor burden and tumor growth in vivo. In conclusion, our results demonstrate how BAMBI downregulation drives the invasiveness of NSCLC, highlighting TGF-beta signaling as a candidate therapeutic target in this setting

Additional file 1: of Aberrant DNA methylation of ADAMTS16 in colorectal and other epithelial cancers

Figure S1. Differentially methylated CpGs in tumor tissue compared to non-tumor tissue in CRC patients. Tumor resectats (canc) and peri-tumoral non-malignant resectats (ctrl) from the same patient were analyzed with the HumanMethylation450 BeadChip Array for the methylation of 450 k CpG sites. 72 of 1145 CpGs located in ADAM/TS genes were differentially methylated. The depicted β-value represents a quantitation of the methylation level of the respective CpG-locus. Data were statistically analyzed with Wilcoxon signed-rank and corrected for multiple testing with Benjamini-Hochberg method (**** P < 0.0001). Hypermethylation was defined as ∆βmean ≥0.2 (P < 0.05) and hypomethylation as ∆βmean ≤ − 0.2 (P < 0.05) compared to the control. Only hyper- or hypomethylated CpGs are presented. p-values were rounded to the 5th decimal place where applicable. The colored bars represent the magnitude of hypermethylation (red), hypomethylation (blue) or the absolute value of the methylation change (green). Figure S2. Methylation status of all ADAMTS16 CpGs in CRC patients. Tumor resectats (n = 117, canc) and peri-tumoral non-malignant tissue (n = 117, ctrl) from the same patient were analyzed with the HumanMethylation450 BeadChip Array for the methylation of 450 k CpG sites. In ADAMTS16, 14 out of 53 CpGs were differentially methylated and 11 CpGs showed intermediate methylation alterations (0.1 ≤ |∆βmean| < 0.2). The depicted β-value represents a quantitation of the methylation level of the respective CpG-locus. Data were statistically analyzed with Wilcoxon signed-rank test and corrected for multiple testing with Benjamini-Hochberg method (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Hypermethylation was defined as ∆βmean ≥0.2 (P < 0.05) and hypomethylation as ∆βmean ≤ − 0.2 (P < 0.05) compared to the control. Ctrl = control, peri-tumoral non-malignant tissue; canc = cancerous tissue. p-values were rounded to the 6th decimal place where applicable. The colored bars represent the magnitude of hypermethylation (red), hypomethylation (blue) or the absolute value of the methylation change (green) (DOCX 491 kb