Kornel Schuebel

Genomic and Epigenomic Integration Identifies a Prognostic Signature in Colon Cancer

Purpose: The importance of genetic and epigenetic alterations maybe in their aggregate role in altering core pathways in tumorigenesis. Experimental Design: Merging genome-wide genomic and epigenomic alterations, we identify key genes and pathways altered in colorectal cancers (CRC). DNA methylation analysis was tested for predicting survival in CRC patients using Cox proportional hazard model. Results: We identified 29 low frequency-mutated genes that are also inactivated by epigenetic mechanisms in CRC. Pathway analysis showed the extracellular matrix (ECM) remodeling pathway is silenced in CRC. Six ECM pathway genes were tested for their prognostic potential in large CRC cohorts (n = 777). DNA methylation of IGFBP3 and EVL predicted for poor survival (IGFBP3: HR = 2.58, 95% CI: 1.37–4.87, P = 0.004; EVL: HR = 2.48, 95% CI: 1.07–5.74, P = 0.034) and simultaneous methylation of multiple genes predicted significantly worse survival (HR = 8.61, 95% CI: 2.16–34.36, P < 0.001 for methylation of IGFBP3, EVL, CD109, and FLNC). DNA methylation of IGFBP3 and EVL was validated as a prognostic marker in an independent contemporary-matched cohort (IGFBP3 HR = 2.06, 95% CI: 1.04–4.09, P = 0.038; EVL HR = 2.23, 95% CI: 1.00–5.0, P = 0.05) and EVL DNA methylation remained significant in a secondary historical validation cohort (HR = 1.41, 95% CI: 1.05–1.89, P = 0.022). Moreover, DNA methylation of selected ECM genes helps to stratify the high-risk stage 2 colon cancers patients who would benefit from adjuvant chemotherapy (HR: 5.85, 95% CI: 2.03–16.83, P = 0.001 for simultaneous methylation of IGFBP3, EVL, and CD109). Conclusions: CRC that have silenced genes in ECM pathway components show worse survival suggesting that our finding provides novel prognostic biomarkers for CRC and reflects the high importance of integrative analyses linking genetic and epigenetic abnormalities with pathway disruption in cancer. Clin Cancer Res; 17(6); 1535–45. ©2011 AACR.

DNA methylation in the medial prefrontal cortex regulates alcohol-induced behavior and plasticity.

Recent studies have suggested an association between alcoholism and DNA methylation, a mechanism that can mediate long-lasting changes in gene transcription. Here, we examined the contribution of DNA methylation to the long-term behavioral and molecular changes induced by a history of alcohol dependence. In search of mechanisms underlying persistent rather than acute dependence-induced neuroadaptations, we studied the role of DNA methylation regulating medial prefrontal cortex (mPFC) gene expression and alcohol-related behaviors in rats 3 weeks into abstinence following alcohol dependence. Postdependent rats showed escalated alcohol intake, which was associated with increased DNA methylation as well as decreased expression of genes encoding synaptic proteins involved in neurotransmitter release in the mPFC. Infusion of the DNA methyltransferase inhibitor RG108 prevented both escalation of alcohol consumption and dependence-induced downregulation of 4 of the 7 transcripts modified in postdependent rats. Specifically, RG108 treatment directly reversed both downregulation of synaptotagmin 2 (Syt2) gene expression and hypermethylation on CpG#5 of its first exon. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in compulsive-like behavior. Our findings identified a functional role of DNA methylation in alcohol dependence-like behavioral phenotypes and a candidate gene network that may mediate its effects. Together, these data provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcoholism.

Local Cues Establish and Maintain Region-Specific Phenotypes of Basal Ganglia Microglia

Microglia play critical roles in tissue homeostasis and can also modulate neuronal function and synaptic connectivity. In contrast to astrocytes and oligodendrocytes, which arise from multiple progenitor pools, microglia arise from yolk sac progenitors and are widely considered to be equivalent throughout the CNS. However, little is known about basic properties of deep brain microglia, such as those within the basal ganglia (BG). Here, we show that microglial anatomical features, lysosome content, membrane properties, and transcriptomes differ significantly across BG nuclei. Region-specific phenotypes of BG microglia emerged during the second postnatal week and were re-established following genetic or pharmacological microglial ablation and repopulation in the adult, indicating that local cues play an ongoing role in shaping microglial diversity. These findings demonstrate that microglia in the healthy brain exhibit a spectrum of distinct functional states and provide a critical foundation for defining microglial contributions to BG circuit function.

Functional Identification of Cancer-Specific Methylation of CDO1, HOXA9, and TAC1 for the Diagnosis of Lung Cancer

Purpose: Non–small cell lung cancer (NSCLC) is the leading cause of cancer mortality in the world. Novel diagnostic biomarkers may augment both existing NSCLC screening methods as well as molecular diagnostic tests of surgical specimens to more accurately stratify and stage candidates for adjuvant chemotherapy. Hypermethylation of CpG islands is a common and important alteration in the transition from normal tissue to cancer. Experimental Design: Following previously validated methods for the discovery of cancer-specific hypermethylation changes, we treated eight NSCLC cell lines with the hypomethylating agent deoxyazacitidine or trichostatin A. We validated the findings using a large publicly available database and two independent cohorts of primary samples. Results: We identified >300 candidate genes. Using The Cancer Genome Atlas (TCGA) and extensive filtering to refine our candidate genes for the greatest ability to distinguish tumor from normal, we define a three-gene panel, CDO1, HOXA9, and TAC1, which we subsequently validate in two independent cohorts of primary NSCLC samples. This three-gene panel is 100% specific, showing no methylation in 75 TCGA normal and seven primary normal samples and is 83% to 99% sensitive for NSCLC depending on the cohort. Conclusion: This degree of sensitivity and specificity may be of high value to diagnose the earliest stages of NSCLC. Addition of this three-gene panel to other previously validated methylation biomarkers holds great promise in both early diagnosis and molecular staging of NSCLC. Clin Cancer Res; 20(7); 1856–64. ©2014 AACR.

Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Aberrant promoter DNA-hypermethylation and repressive chromatin constitutes a frequent mechanism of gene inactivation in cancer. There is great interest in dissecting the mechanisms underlying this abnormal silencing. Studies have shown changes in the nuclear organization of chromatin in tumor cells as well as the association of aberrant methylation with long-range silencing of neighboring genes. Furthermore, certain tumors show a high incidence of promoter methylation termed as the CpG island methylator phenotype. Here, we have analyzed the role of nuclear chromatin architecture for genes in hypermethylated inactive versus nonmethylated active states and its relation with long-range silencing and CpG island methylator phenotype. Using combined immunostaining for active/repressive chromatin marks and fluorescence in situ hybridization in colorectal cancer cell lines, we show that aberrant silencing of these genes occurs without requirement for their being positioned at heterochromatic domains. Importantly, hypermethylation, even when associated with long-range epigenetic silencing of neighboring genes, occurs independent of their euchromatic or heterochromatic location. Together, these results indicate that, in cancer, extensive changes around promoter chromatin of individual genes or gene clusters could potentially occur locally without preference for nuclear position and/or causing repositioning. These findings have important implications for understanding relationships between nuclear organization and gene expression patterns in cancer.

Making Sense of Epigenetics

The gene-environment interactions that underlie development and progression of psychiatric illness are poorly understood. Despite a century of progress, genetic approaches have failed to identify new treatment modalities, perhaps because of the heterogeneity of the disorders and lack of understanding of mechanisms. Recent exploration into epigenetic mechanisms in health and disease has uncovered changes in DNA methylation and chromatin structure that may contribute to psychiatric disorders. Epigenetic changes suggest a variety of new therapeutic options due to their reversible chemistry. However, distinguishing causal links between epigenetic changes and disease from changes consequent to life experience has remained problematic. Here we define epigenetics and explore aspects of epigenetics relevant to causes and mechanisms of psychiatric disease, and speculate on future directions.

A Four-Gene Promoter Methylation Marker Panel Consisting of GREM1, NEURL, LAD1, and NEFH Predicts Survival of Clear Cell Renal Cell Cancer Patients

Purpose: The currently used prognostic models for patients with nonmetastatic clear cell renal cell carcinoma (ccRCC) are based on clinicopathologic features and might be improved by adding molecular markers. Epigenetic alterations occur frequently in ccRCC and are promising biomarkers. The aim of this study is to identify prognostic promoter methylation markers for ccRCC. Experimental Design: We integrated data generated by massive parallel sequencing of methyl-binding domain enriched DNA and microarray-based RNA expression profiling of 5-aza-2′-deoxycytidine–treated ccRCC cell lines to comprehensively characterize the ccRCC methylome. A selection of the identified methylation markers was evaluated in two independent series of primary ccRCC (n = 150 and n = 185) by methylation-specific PCR. Kaplan–Meier curves and log-rank tests were used to estimate cause-specific survival. HRs and corresponding 95% confidence intervals (CI) were assessed using Cox proportional hazard models. To assess the predictive capacity and fit of models combining several methylation markers, HarrellC statistic and the Akaike Information Criterion were used. Results: We identified four methylation markers, that is, GREM1, NEURL, LAD1, and NEFH, that individually predicted prognosis of patients with ccRCC. The four markers combined were associated with poorer survival in two independent patient series (HR, 3.64; 95% CI, 1.02–13.00 and HR, 7.54; 95% CI, 2.68–21.19). These findings were confirmed in a third series of ccRCC cases from The Cancer Genome Atlas (HR, 3.60; 95% CI, 2.02–6.40). Conclusions: A four-gene promoter methylation marker panel consisting of GREM1, NEURL, LAD1, and NEFH predicts outcome of patients with ccRCC and might be used to improve current prognostic models. Clin Cancer Res; 23(8); 2006–18. ©2016 AACR.

Spectrin Repeat Containing Nuclear Envelope 1 and Forkhead Box Protein E1 Are Promising Markers for the Detection of Colorectal Cancer in Blood

Identifying biomarkers in body fluids may improve the noninvasive detection of colorectal cancer. Previously, we identified N-Myc downstream-regulated gene 4 (NDRG4) and GATA binding protein 5 (GATA5) methylation as promising biomarkers for colorectal cancer in stool DNA. Here, we examined the utility of NDRG4, GATA5, and two additional markers [Forkhead box protein E1 (FOXE1) and spectrin repeat containing nuclear envelope 1 (SYNE1)] promoter methylation as biomarkers in plasma DNA. Quantitative methylation-specific PCR was performed on plasma DNA from 220 patients with colorectal cancer and 684 noncancer controls, divided in a training set and a test set. Receiver operating characteristic analysis was performed to measure the area under the curve of GATA5, NDRG4, SYNE1, and FOXE1 methylation. Functional assays were performed in SYNE1 and FOXE1 stably transfected cell lines. The sensitivity of NDRG4, GATA5, FOXE1, and SYNE1 methylation in all stages of colorectal cancer (154 cases, 444 controls) was 27% [95% confidence interval (CI), 20%–34%), 18% (95% CI, 12%–24%), 46% (95% CI, 38%–54%), and 47% (95% CI, 39%–55%), with a specificity of 95% (95% CI, 93%–97%), 99% (95% CI, 98%–100%), 93% (95% CI, 91%–95%), and 96% (95% CI, 94%–98%), respectively. Combining SYNE1 and FOXE1, increased the sensitivity to 56% (95% CI, 48%–64%), while the specificity decreased to 90% (95% CI, 87%–93%) in the training set and to 58% sensitivity (95% CI, 46%–70%) and 91% specificity (95% CI, 80%–100%) in a test set (66 cases, 240 controls). SYNE1 overexpression showed no major differences in cell proliferation, migration, and invasion compared with controls. Overexpression of FOXE1 significantly decreased the number of colonies in SW480 and HCT116 cell lines. Overall, our data suggest that SYNE1 and FOXE1 are promising markers for colorectal cancer detection. Cancer Prev Res; 8(2); 157–64. ©2014 AACR.

Timeless insights into prevention of acetaldehyde genotoxicity

The International Agency for Research on Cancer classified acetaldehyde related to alcoholic beverage consumption as carcinogenic to humans. This classification was based in large part on epidemiological studies of individuals who lack the capacity to metabolize acetaldehyde due to a functional polymorphism in ALDH2. Such individuals have a dramatically elevated risk of esophageal cancer from alcohol consumption than those with fully active ALDH2. Acetaldehyde forms DNA adducts supporting a genotoxic mechanism of carcinogenicity. To identify molecular mechanisms that protect against acetaldehyde genotoxicity, Noguchi et al. performed a comprehensive mutational analysis of the role of the DNA repair and DNA damage response pathways, using the fission yeast Schizosaccharomyces pombe as a model organism. The experimental design involved exposing wild-type and mutant strains to increasing concentrations of acetaldehyde, then monitoring cell growth as an end point. In some experiments Rad52-YFP was used as a marker of DNA damage. Rad52 is a DNA recombinase that is recruited to sites of DNA damage, forming DNA repair foci. Homology searches identified 3 possible yeast Aldh genes, denoted Atd1-3. Of these, Atd1 was found to play the major role in protection against acetaldehyde, though cells lacking either Atd1 or Atd2 show elevated levels of DNA damage in response to acetaldehyde. In Atd1D cells, deletion of the replication fork protection complex protein Swi1 increased sensitivity to acetaldehyde, and also resulted in a dramatic decrease in chromosome number following acetaldehyde exposure. Swi1 forms a complex with Swi3 that is homologous to the TimelessTipin complex in human cells. In cells with wild-type Atd1, deletion of both Swi1 and rad26 resulted in a much greater acetaldehyde sensitive than either single mutant. The rad26 gene encodes a protein that interacts with the cell cycle checkpoint kinase Rad3, homologous to ATR in mammals. Consistent with work in mammalian cells ( and references therein), disruption of either nucleotide excision repair (NER), the Fanconi anemia DNA damage response pathway, or homologous recombination repair increased sensitivity to acetaldehyde, as did disruption of multiple translesion synthesis DNA polymerases. Interestingly, acetaldehyde sensitivity was also increased in cells with mutations in in either AP-endonuclease 2 (apn2 D) or Endonuclease III (nth1 D), consistent with a role for base excision repair (BER) in protecting against acetaldehyde toxicity. This observation is somewhat surprising, since acetaldehyde-DNA lesions are not known to be repaired by BER. However, exposure of living cells to acetaldehyde can result in ethenobase adducts, via an indirect pathway involving lipid peroxidation; some of these adducts are substrates for BER. It is worth noting however that apn2 D cells and nth1 D cells were only sensitive to acetaldehyde in the context of a cell cycle checkpoint defect (rad3 D). Another notable finding from this work is evidence of important roles for Wss-related proteases in protecting against acetaldehyde toxicity. The Wss-related proteases had been implicated in proteolysis related to the repair of DNA – protein crosslinks, which can result from acetaldehyde. These proteases are homologous to the human DVC1-Spartan protease. Interestingly, germline mutations in the DVC1-Spartan gene in humans results in premature aging and an increased risk of liver cancer. A summary of the main pathways for protecting against acetaldehyde genotoxicity identified by Noguchi et al. is given in Fig. 1. One limitation of the current work is that it relied heavily on measures of relative cell growth of different mutant strains under increasing concentrations of acetaldehyde. While this approach is certainly reasonable for rapid screening of many single and multiple mutant strains, future studies could include assays of specific mutagenic events, which might be observed at lower acetaldehyde concentrations, or in strains containing single gene mutations that appeared normal using cell growth assays. To assess the relevance of these findings for human disease, it will be important to validate these findings in human cells, under acetaldehyde concentrations that are relevant to human exposures. This can be done in a straightforward manner by knocking down the relevant human genes, and assessing effects

Ventral midbrain astrocytes display unique physiological features and sensitivity to dopamine D2 receptor signaling

Astrocytes are ubiquitous CNS cells that support tissue homeostasis through ion buffering, neurotransmitter recycling, and regulation of CNS vasculature. Yet, despite the essential functional roles they fill, very little is known about the physiology of astrocytes in the ventral midbrain, a region that houses dopamine-releasing neurons and is critical for reward learning and motivated behaviors. Here, using a combination of whole-transcriptome sequencing, histology, slice electrophysiology, and calcium imaging, we performed the first functional and molecular profiling of ventral midbrain astrocytes and observed numerous differences between these cells and their telencephalic counterparts, both in their gene expression profile and in their physiological properties. Ventral midbrain astrocytes have very low membrane resistance and inward-rectifying potassium channel-mediated current, and are extensively coupled to surrounding oligodendrocytes through gap junctions. They exhibit calcium responses to glutamate but are relatively insensitive to norepinephrine. In addition, their calcium activity can be dynamically modulated by dopamine D2 receptor signaling. Taken together, these data indicate that ventral midbrain astrocytes are physiologically distinct from astrocytes in cortex and hippocampus. This work provides new insights into the extent of functional astrocyte heterogeneity within the adult brain and establishes the foundation for examining the impact of regional astrocyte differences on dopamine neuron function and susceptibility to degeneration.