The epigenome is defined by DNA methylation patterns and the associated posttranslational modifications of histones. This histone code determines the expression status of individual genes dependent upon their localization on the chromatin. The silencing of gene expression is associated with deacetylated histones, which are often found to be associated with regions of DNA methylation as well as methylation at the lysine 4 residue of histone 3. In contrast, the activation of gene expression is associated with acetylated histones and methylation at the lysine 9 residue of histone 3. The histone deactylases play a major role in keeping the balance between the acetylated and deacetylated states of chromatin. Histone deacetylases (HDACs) are divided into three classes: class I HDACs (HDACs 1, 2, 3, and 8) are similar to the yeast RPD3 protein and localize to the nucleus; class II HDACs (HDACs 4, 5, 6, 7, 9, and 10) are homologous to the yeast HDA1 protein and are found in both the nucleus and cytoplasm; and class III HDACs form a structurally distinct class of NAD-dependent enzymes that are similar to the yeast SIR2 proteins. Since inappropriate silencing of critical genes can result in one or both hits of tumor suppressor gene (TSG) inactivation in cancer, theoretically the reactivation of affected TSGs could have an enormous therapeutic value in preventing and treating cancer. Indeed, several HDAC inhibitors are currently being developed and tested for their potency in cancer chemotherapy. Importantly, these agents are also potentially applicable to chemoprevention if their toxicity can be minimized. Despite the toxic side effects and lack of specificity of some of the inhibitors, progress is being made. With the elucidation of the structures, functions and modes of action of HDACs, finding agents that may be targeted to specific HDACs and potentially reactivate expression of only a defined set of affected genes in cancer will be more attainable.

Breast cancer progression is associated with aberrant DNA methylation and expression of genes that control the epithelial-mesenchymal transition (EMT), a critical step in malignant conversion. Although the genes affected have been studied, there is little understanding of how aberrant activation of the DNA methylation machinery itself occurs. Using a breast cancer cell-based model system, we found that cells that underwent EMT exhibited overactive transforming growth factor β (TGFβ) signaling and loss of expression of the CDH1, CGN, CLDN4, and KLK10 genes as a result of hypermethylation of their corresponding promoter regions. Based on these observations, we hypothesized that activated TGFβ-Smad signaling provides an "epigenetic memory" to maintain silencing of critical genes. In support of this hypothesis, disrupting Smad signaling in mesenchymal breast cancer cells resulted in DNA demethylation and reexpression of the genes identified. This epigenetic reversal was accompanied by an acquisition of epithelial morphology and a suppression of invasive properties. Notably, disrupting TGFβ signaling decreased the DNA binding activity of DNA methyltransferase DNMT1, suggesting that failure to maintain methylation of newly synthesized DNA was the likely cause of DNA demethylation. Together, our findings reveal a hyperactive TGFβ-TGFβR-Smad2 signaling axis needed to maintain epigenetic silencing of critical EMT genes and breast cancer progression. Cancer Res; 70(3); 968-78. ©2010 AACR.

Metastatic dissemination of breast cancer cells represents a significant clinical obstacle to curative therapy. The loss of function of metastasis suppressor genes is a major rate-limiting step in breast cancer progression that prevents the formation of new colonies at distal sites. However, the discovery of new metastasis suppressor genes in breast cancer using genomic efforts has been slow, potentially due to their primary regulation by epigenetic mechanisms. Here, we report the use of model cell lines with the same genetic lineage for the identification of a novel metastasis suppressor gene, serum deprivation response (SDPR), localized to 2q32-33, a region reported to be associated with significant loss of heterozygosity in breast cancer. In silico metaanalysis of publicly available gene expression datasets suggests that the loss of expression of SDPR correlates with significantly reduced distant-metastasis–free and relapse-free survival of breast cancer patients who underwent therapy. Furthermore, we found that stable SDPR overexpression in highly metastatic breast cancer model cell lines inhibited prosurvival pathways, shifted the balance of Bcl-2 family proteins in favor of apoptosis, and decreased migration and intravasation/extravasation potential, with a corresponding drastic suppression of metastatic nodule formation in the lungs of NOD/SCID mice. Moreover, SDPR expression is silenced by promoter DNA methylation, and as such it exemplifies epigenetic regulation of metastatic breast cancer progression. These observations highlight SDPR as a potential prognostic biomarker and a target for future therapeutic applications.

Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non^small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fisher's exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fisher's exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer^associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.Lung cancer is the leading cause of cancer deaths in the United States, accounting for 28% of all cancer deaths and f157,200 deaths every year (1). Unfortunately, 36% of nonsmall cell lung cancer cases are detected at an advanced stage often after micrometastasis has developed, leading to an alarmingly low 5-year survival rate of only 14.9% (1). Because nearly 87% of lung cancer cases are due to smoking, yet only 10% of smokers develop lung cancer, it may be worthwhile to screen for susceptible individuals to administer effective preventive measures (1, 2). Thus, the establishment of a combination of early diagnostic markers that could be analyzed in clinical samples obtained using relatively noninvasive procedures could become an asset to efficient detection of changes in preneoplastic tissue before tumor formation and metastasis can occur.Differential DNA methylation at CpG islands has been associated with regulation of gene expression and is essential for normal development, X-chromosome inactivation, imprinting, suppression of parasitic DNA sequences, and cancer (3 -5). Aberrant differential methylation of CpG islands in the promoter region of genes that are implicated in different roles including carcinogen activation or detoxification (CYP1A1 and GSTP1), tumor suppression (p14, p15, p16, p73, APC, and BRCA1), DNA repair (hMLH1 and MGMT), and metastasis and invasion (CDH1, ECAD, TIMP1, and DAPK) occurs in several cancers, including lung cancer (3 -10). Thus, the DNA methylation status of critical genes is not only ideal for use as diagnostic markers but also as therapeutic targets for lung cancer. In this study, we chose to a...

IntroductionBasal-like breast cancer (BLBC) is an aggressive subtype often characterized by distant metastasis, poor patient prognosis, and limited treatment options. Therefore, the discovery of alternative targets to restrain its metastatic potential is urgently needed. In this study, we aimed to identify novel genes that drive metastasis of BLBC and to elucidate the underlying mechanisms of action.MethodsAn unbiased approach using gene expression profiling of a BLBC progression model and in silico leveraging of pre-existing tumor transcriptomes were used to uncover metastasis-promoting genes. Lentiviral-mediated knockdown of interleukin-13 receptor alpha 2 (IL13Ralpha2) coupled with whole-body in vivo bioluminescence imaging was performed to assess its role in regulating breast cancer tumor growth and lung metastasis. Gene expression microarray analysis was followed by in vitro validation and cell migration assays to elucidate the downstream molecular pathways involved in this process.ResultsWe found that overexpression of the decoy receptor IL13Ralpha2 is significantly enriched in basal compared with luminal primary breast tumors as well as in a subset of metastatic basal-B breast cancer cells. Importantly, breast cancer patients with high-grade tumors and increased IL13Ralpha2 levels had significantly worse prognosis for metastasis-free survival compared with patients with low expression. Depletion of IL13Ralpha2 in metastatic breast cancer cells modestly delayed primary tumor growth but dramatically suppressed lung metastasis in vivo. Furthermore, IL13Ralpha2 silencing was associated with enhanced IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and impaired migratory ability of metastatic breast cancer cells. Interestingly, genome-wide transcriptional analysis revealed that IL13Ralpha2 knockdown and IL-13 treatment cooperatively upregulated the metastasis suppressor tumor protein 63 (TP63) in a STAT6-dependent manner. These observations are consistent with increased metastasis-free survival of breast cancer patients with high levels of TP63 and STAT6 expression and suggest that the STAT6-TP63 pathway could be involved in impairing metastatic dissemination of breast cancer cells to the lungs.ConclusionOur findings indicate that IL13Ralpha2 could be used as a promising biomarker to predict patient outcome and provide a rationale for assessing the efficacy of anti-IL13Ralpha2 therapies in a subset of highly aggressive basal-like breast tumors as a strategy to prevent metastatic disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0607-y) contains supplementary material, which is available to authorized users.

A correlation between reduction in 5-HTT expression and DNA hypermethylation of the 5-HTT promoter in drug naïve SCZ patients suggests that an epigenetically defined hypo-activity of 5-HTT may be linked to SCZ pathogenesis. Furthermore, this epigenetic mark in DNA extracted from saliva can be considered as one of the key determinants in a panel of diagnostic and/or therapeutic biomarkers for SCZ.

High frequency of chromosomal deletions elicited as losses of heterozygosity is a hallmark of genomic instability in cancer. Functional losses of tumor suppressor genes caused by loss of heterozygosity at defined regions during clonal selection for growth advantage define the minimally lost regions as their likely locations on chromosomes. Loss of heterozygosity is elicited at the molecular or cytogenetic level as a deletion, a gene conversion, single or double homologous and nonhomologous mitotic recombinations, a translocation, chromosome breakage and loss, chromosomal fusion or telomeric end-to-end fusions, or whole chromosome loss with or without accompanying duplication of the retained chromosome. Because of the high level of specificity, loss of heterozygosity has recently become invaluable as a marker for diagnosis and prognosis of cancer. The molecular defects for the occurrence of loss of heterozygosity are derived from disabled caretaker genes, which protect the integrity of DNA, or chromosome segregator genes, which mediate faithful chromosome disjunction.

Due to the lack of genetic association between individual genes and schizophrenia (SCZ) pathogenesis, the current consensus is to consider both genetic and epigenetic alterations. Here, we report the examination of DNA methylation status of DTNBP1 promoter region, one of the most credible candidate genes affected in SCZ, assayed in saliva and post-mortem brain samples. The Illumina DNA methylation profiling and bisulfite sequencing of representative samples were used to identify methylation status of the DTNBP1 promoter region. Quantitative methylation specific PCR (qMSP) was employed to assess methylation of DTNBP1 promoter CpGs flanking a SP1 binding site in the saliva of SCZ patients, their first-degree relatives and control subjects (30, 15, and 30/group, respectively) as well as in post-mortem brains of patients with SCZ and bipolar disorder (BD) versus controls (35/group). qRT-PCR was used to assess DTNBP1 expression. We found DNA hypermethylation of DTNBP1 promoter in the saliva of SCZ patients (∼12.5%, P = 0.036), particularly in drug-naïve patients (∼20%, P = 0.011), and a trend toward hypermethylation in their first-degree relatives (P = 0.085) versus controls. Analysis of post-mortem brain samples revealed an inverse correlation between DTNBP1 methylation and expression, and normalization of this epigenetic change by classic antipsychotic drugs. Additionally, BD patients with psychotic depression exhibited higher degree of methylation versus other BD patients (∼80%, P = 0.025). DTNBP1 promoter DNA methylation may become a key element in a panel of biomarkers for diagnosis, prevention, or therapy in SCZ and at risk individuals pending confirmatory studies with larger sample sizes to attain a higher degree of significance.