Fountain of youth for squamous cell carcinomas? On the epigenetic age of non-small cell lung cancer and corresponding tumor-free lung tissues.

Aging affects the core processes of almost every organism, and the functional decline at the cellular and tissue levels influences disease development. Recently, it was shown that the methylation of certain CpG dinucleotides correlates with chronological age and that this epigenetic clock can be applied to study aging-related effects. We investigated these molecular age loci in non-small cell lung cancer (NSCLC) tissues from patients with adenocarcinomas (AC) and squamous cell carcinomas (SQC) as well as in matched tumor-free lung tissue. In both NSCLC subtypes, the calculated epigenetic age did not correlate with the chronological age. In particular, SQC exhibited rejuvenation compared to the corresponding normal lung tissue as well as with the chronological age of the donor. Moreover, the younger epigenetic pattern was associated with a trend toward stem cell-like gene expression patterns. These findings show deep phenotypic differences between the tumor entities AC and SQC, which might be useful for novel therapeutic and diagnostic approaches.

Aberrant DNA methylation of ADAMTS16 in colorectal and other epithelial cancers.

BACKGROUND: ADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors. METHODS: We investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry. RESULTS: In CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation. CONCLUSIONS: We identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.

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.

Epigenetic modifications of the immune-checkpoint genes CTLA4 and PDCD1 in non-small cell lung cancer results in increased expression.

Targeting checkpoint inhibitors using monoclonal antibodies results in significantly better outcome of cancer patients compared to conventional chemotherapy. However, the current companion diagnostics to predict response is so far suboptimal, since they base on more or less reliable immunohistochemical approaches. In order to overcome these limitations, we analyzed epigenetic modifications of PDCD1 (PD1), CD274 (PD-L1), and CTLA4 in NSCLC tissues from 39 patients. Results were correlated with transcriptome data. Significant differences in the CpG-methylation patterns between tumor tissues and matched controls were observed for CTLA4 and PDCD1 (PD1) showing a decreased methylation of these genes compared to matched tumor-free tissues from the same patients. Results were confirmed by bisulfide sequencing in an independent validation cohort. Hypomethylation also resulted in increased expression of these genes as shown by transcriptome data. These epigenetic pathways as a hallmark of NSCLC might be useful to generate more precise diagnostic approaches in the future.

Epigenetic modifications of the VGF gene in human non-small cell lung cancer tissues pave the way towards enhanced expression.

Hwang et al. recently showed that VGF substantially contributes to the resistance of human lung cancer cells towards epidermal growth factor receptor kinase inhibitors. This was further linked to enhanced epithelial-mesenchymal transition. Here, we demonstrate that VGF is epigenetically modified in non-small cell lung cancer tissues compared to corresponding tumor-free lung tissues from the same donors by using methylome bead chip analyses. These epigenetic modifications trigger an increased transcription of the VGF gene within the tumors, which then leads to an increased expression of the protein, facilitating epithelial-mesenchymal transition, and the resistance to kinase inhibitors. These results should be taken into account in the design of novel therapeutic and diagnostic approaches.

Downregulation of the TGFß Pseudoreceptor BAMBI in Non-Small Cell Lung Cancer Enhances TGFß 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 TGFß 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 TGFß signaling pathway. Alterations of epithelial-to-mesenchymal transition (EMT) marker expression were observed in lung cancer samples compared with tumor-free tissues. Distinct alterations in the DNA methylation of the gene regions encoding TGFß pathway components were detected in NSCLC samples compared with 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 TGFß-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 TGFß signaling as a candidate therapeutic target in this setting. Cancer Res; 76(13); 3785-801. ©2016 AACR.