Lung cancer is the leading cause of cancer death worldwide, and adenocarcinoma is its most common histological subtype. Clinical and molecular evidence indicates that lung adenocarcinoma is a heterogeneous disease, which has important implications for treatment. Here we performed genome-scale DNA methylation profiling using the Illumina Infinium HumanMethylation27 platform on 59 matched lung adenocarcinoma/non-tumor lung pairs, with genome-scale verification on an independent set of tissues. We identified 766 genes showing altered DNA methylation between tumors and non-tumor lung. By integrating DNA methylation and mRNA expression data, we identified 164 hypermethylated genes showing concurrent down-regulation, and 57 hypomethylated genes showing increased expression. Integrated pathways analysis indicates that these genes are involved in cell differentiation, epithelial to mesenchymal transition, RAS and WNT signaling pathways, and cell cycle regulation, among others. Comparison of DNA methylation profiles between lung adenocarcinomas of current and never-smokers showed modest differences, identifying only LGALS4 as significantly hypermethylated and down-regulated in smokers.LGALS4, encoding a galactoside-binding protein involved in cell-cell and cell-matrix interactions, was recently shown to be a tumor suppressor in colorectal cancer. Unsupervised analysis of the DNA methylation data identified two tumor subgroups, one of which showed increased DNA methylation and was significantly associated with KRAS mutation and to a lesser extent, with smoking. Our analysis lays the groundwork for further molecular studies of lung adenocarcinoma by identifying novel epigenetically deregulated genes potentially involved in lung adenocarcinoma development/progression, and by describing an epigenetic subgroup of lung adenocarcinoma associated with characteristic molecular alterations.

Carcinoma of the lung is the most common cause of cancer death worldwide. The estimated 5-year survival ranges from 6 -16%, depending on the cell type. The best opportunity for improving survival of lung cancer patients is through early detection, when curative surgical resection is possible. Although the subjects at increased risk for developing carcinoma of the lung (long-term smokers) can be identified, only 10 -20% of this group will ultimately develop the disease. Screening tests of long-term smokers employed to date (radiography and sputum cytology) have not been successful in reducing lung cancer mortality. The application of molecular markers specific for lung cancer offers new possibilities for early detection. Hypermethylation of CpG islands in the promoter regions of genes is a common phenomenon in lung cancer, as demonstrated by the analysis of the methylation status of over 40 genes from lung cancer tumors, cell lines, patient sputum and/ or serum. Determination of the methylation patterns of multiple genes to obtain complex DNA methylation signatures promises to provide a highly sensitive and specific tool for lung cancer diagnosis. When combined with the development of non-invasive methods to detect such signatures, this may provide a viable method to screen subjects at risk for lung cancer.

The RNA-binding protein HuR stabilizes labile mRNAs carrying AU-rich instability elements. This mRNA stabilization can be induced by hypoxia, lipopolysaccharide, and UV light. The mechanism by which these stimuli activate HuR is unclear and might be related to post-translational modification of this protein.Here we show that HuR can be methylated on arginine. However, HuR is not a substrate for PRMT1, the most prominent protein-arginine methyltransferase in mammalian cells, which methylates a number of heterogeneous nuclear ribonucleoproteins. Instead, HuR is specifically methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase previously shown to serve as a transcriptional coactivator. By analyzing methylation of specific HuR arginine-to-lysine mutants and by sequencing radioactively methylated HuR peptides, Arg 217 was identified as the major HuR methylation site. Arg 217 is located in the hinge region between the second and third of the three HuR RNA recognition motif domains. Antibodies against a methylated HuR peptide were used to demonstrate in vivo methylation of HuR. HuR methylation increased in cells that overexpressed CARM1. Importantly, lipopolysaccharide stimulation of macrophages, which leads to HuR-mediated stabilization of tumor necrosis factor ␣ mRNA in these cells, caused increased methylation of endogenous HuR. Thus, CARM1, which plays a role in transcriptional activation through histone H3 methylation, may also play a role in post-transcriptional gene regulation by methylating HuR.

Epithelial to mesenchymal transition (EMT) is a key process associated with tumor progression and metastasis. To define molecular features associated with EMT states, we undertook an integrative approach combining mRNA, microRNA, DNA methylation and proteomic profiles of 38 cell populations representative of the genomic heterogeneity in lung adenocarcinoma were integrated with functional profiles consisting of cell invasiveness, adhesion and motility. A subset of cell lines that were readily defined as epithelial or mesenchymal based on their morphology and E-cadherin and vimentin expression elicited distinctive molecular signatures. However, most cell populations displayed intermediate/hybrid states of EMT, with mixed epithelial and mesenchymal characteristics. A dominant proteomic feature of aggressive hybrid cell lines was upregulation of cytoskeletal and actin binding proteins, a signature shared with mesenchymal cell lines. Cytoskeletal reorganization preceded loss of E-cadherin in epithelial cells in which EMT was induced by TGFβ. A set of transcripts corresponding to the mesenchymal protein signature enriched in cytoskeletal proteins was found to be predictive of survival in independent datasets of lung adenocarcinomas. Our findings point to an association between cytoskeletal and actin-binding proteins, a mesenchymal or hybrid EMT phenotype and invasive properties of lung adenocarcinomas.

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters, and CpG islands (CGIs), while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, 4 of the 5 established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome.