Pancreatic cancer is the fourth leading cause of cancer deaths and there currently is no reliable modality for the early detection of this disease. Here we identify cancer-specific promoter DNA methylation of BNC1 and ADAMTS1 as a promising biomarker detection strategy meriting investigation in pancreatic cancer.
We used a genome-wide pharmacologic transcriptome approach to identify novel cancer-specific DNA methylation alterations in pancreatic cancer cell lines. Of 8 promising genes, we focused our studies on BNC1 and ADAMTS1 for further downstream analysis including methylation and expression. We used a nanoparticle-enabled MOB (Methylation On Beads) technology to detect early stage pancreatic cancers by analyzing DNA methylation in patient serum.
We identified 2 novel genes, BNC1 (92%) and ADAMTS1, (68%) that showed a high frequency of methylation in pancreas cancers (n=143), up to 100% in PanIN-3 and 97% in Stage I invasive cancers. Using the nanoparticle-enabled MOB technology, these alterations could be detected in serum samples (n=42) from pancreas cancer patients, with a sensitivity for BNC1 of 79% (95%CI:66-91%) and for ADAMTS1 of 48% (95%CI:33-63%), while specificity was 89% for BNC1 (95%CI:76-100%) and 92% for ADAMTS1 (95%CI:82-100%). Overall sensitivity using both markers is 81% (95%CI:69-93%) and specificity is 85% (95%CI:71-99%).
Promoter DNA methylation of BNC1 and ADAMTS1 are potential biomarkers to detect early stage pancreatic cancers. Assaying the promoter methylation status of these genes in circulating DNA from serum is a promising strategy for early detection of pancreatic cancer and has the potential to improve mortality from this disease.
DNA Methylation; Early detection biomarker; Pancreatic cancer; Cancer screening
Epigenetic therapies may play a prominent role in the future management of solid tumors. This possibility is based on the clinical efficacy of existing drugs in treating defined hematopoietic neoplasms, paired with promising new data from preclinical and clinical studies that examined these agents in solid tumors. We suggest that current drugs may represent a targeted therapeutic approach for reprogramming solid tumor cells, a strategy that must be pursued in concert with the explosion in knowledge about the molecular underpinnings of normal and cancer epigenomes. We hypothesize that understanding targeted proteins in the context of their enzymatic and scaffolding functions and in terms of their interactions in complexes with proteins that are targets of new drugs under development defines the future of epigenetic therapies for cancer.
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.
Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also knownasPD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.
Innovative therapies are needed for advanced Non-Small Cell Lung Cancer (NSCLC). We have undertaken a genomics based, hypothesis driving, approach to query an emerging potential that epigenetic therapy may sensitize to immune checkpoint therapy targeting PD-L1/PD-1 interaction. NSCLC cell lines were treated with the DNA hypomethylating agent azacytidine (AZA – Vidaza) and genes and pathways altered were mapped by genome-wide expression and DNA methylation analyses. AZA-induced pathways were analyzed in The Cancer Genome Atlas (TCGA) project by mapping the derived gene signatures in hundreds of lung adeno (LUAD) and squamous cell carcinoma (LUSC) samples. AZA up-regulates genes and pathways related to both innate and adaptive immunity and genes related to immune evasion in a several NSCLC lines. DNA hypermethylation and low expression of IRF7, an interferon transcription factor, tracks with this signature particularly in LUSC. In concert with these events, AZA up-regulates PD-L1 transcripts and protein, a key ligand-mediator of immune tolerance. Analysis of TCGA samples demonstrates that a significant proportion of primary NSCLC have low expression of AZA-induced immune genes, including PD-L1. We hypothesize that epigenetic therapy combined with blockade of immune checkpoints – in particular the PD-1/PD-L1 pathway – may augment response of NSCLC by shifting the balance between immune activation and immune inhibition, particularly in a subset of NSCLC with low expression of these pathways. Our studies define a biomarker strategy for response in a recently initiated trial to examine the potential of epigenetic therapy to sensitize patients with NSCLC to PD-1 immune checkpoint blockade.
Non-Small Cell Lung Cancer(NSCLC); Azacytidine; HDAC inhibitor
Epigenetic abnormalities in lung and other cancers continue to be defined at a rapid pace. We are coming to appreciate that cancers have an “epigenetic landscape” wherein genes vulnerable to abnormalities, such as promoter DNA hypermethylation and associated gene silencing, tend to reside in defined nuclear positions and chromosome domains and relationships to chromatin regulation, which facilitates states of stem cell renewal. These same genes and domains are also vulnerable to epigenetic abnormalities induced by factors to which cells are exposed during cancer risk states, such as chronic inflammation. We can use all of this basic information for translational purposes in terms of deriving biomarkers for cancer risk states and detection and therapeutic strategies.
epigenetic; cancer; DNA hypermethylation
Reversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower, less toxic, doses. Experimentally, high doses induce rapid DNA damage and cytotoxicity, which do not explain the prolonged response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically-relevant nanomolar doses, without causing immediate cytotoxicity, produce an anti-tumor “memory” response, including inhibition of subpopulations of cancer stem-like cells. These effects are accompanied by sustained decreases in genome-wide promoter DNA methylation, gene re-expression, and anti-tumor changes in key cellular regulatory pathways. Low dose decitabine and azacitidine may have broad applicability for cancer management.
In this article we review many important epigenetic changes in early carcinogenesis and discuss the possibility of these alterations being targeted for therapeutic intervention in the future. Both regional DNA methylation and global chromatin packaging are interrelated partners that function in concert to control gene transcription. We first summarize briefly DNA methylation and its role in gene expression. Then, we focus on how the DNA is packaged into chromatin and the tight relationship between chromatin and DNA methylation. A more complete understanding of these key, regulatory events is vital in approaching a more rational drug therapy to various malignancies.
cancer; chromatin modification; DNA methylation; epigenetics
Genome-wide DNA methylation analyses have identified hundreds of candidate DNA-hypermethylated genes in cancer. Comprehensive functional analyses provide an understanding of the biologic significance of this vast amount of DNA methylation data that may allow the determination of key epigenetic events associated with tumorigenesis.
To study mechanisms of cysteine dioxygenase type 1 (CDO1) inactivation and its functional significance in breast cancer in a comprehensive manner, we screened for DNA methylation and gene mutations in primary breast cancers and analyzed growth, survival, and reactive oxygen species (ROS) production in breast cancer cells with restored CDO1 function in the context of anthracycline treatment.
DNA methylation-associated silencing of CDO1 in breast cancer is frequent (60%), cancer specific, and correlates with disease progression and outcome. CDO1 function can alternatively be silenced by repressive chromatin, and we describe protein-damaging missense mutations in 7% of tumors without DNA methylation. Restoration of CDO1 function in breast cancer cells increases levels of ROS and leads to reduced viability and growth, as well as sensitization to anthracycline treatment. Priming with 5-azacytidine of breast cancer cells with epigenetically silenced CDO1 resulted in restored expression and increased sensitivity to anthracyclines.
We report that silencing of CDO1 is a critical epigenetic event that contributes to the survival of oxidative-stressed breast cancer cells through increased detoxification of ROS and thus leads to the resistance to ROS-generating chemotherapeutics including anthracyclines. Our study shows the importance of CDO1 inactivation in breast cancer and its clinical potential as a biomarker and therapeutic target to overcome resistance to anthracyclines.
WNT signaling pathway dysregulation is an important event in the pathogenesis of colorectal cancer (CRC) with APC mutations seen in more than 80% of sporadic CRC. However, such mutations in the WNT signaling pathway genes are rare in inflammatory bowel disease (IBD) associated neoplasia (dysplasia and cancer). This study examined the role of epigenetic silencing of WNT signaling pathway genes in the pathogenesis of IBD-associated neoplasia.
Paraffin-embedded tissue samples were obtained and methylation of ten WNT signaling pathway genes, including APC1A, APC2, SFRP1, SFRP2, SFRP4, SFRP5, DKK1, DKK3, WIF1 and LKB1, was analyzed. Methylation analysis was performed on 41 IBD samples, 27 normal colon samples (NCs), and 24 sporadic CRC samples.
Methylation of WNT signaling pathway genes is a frequent and early event in IBD and IBD-associated neoplasia. A progressive increase in the percentage of methylated genes in the WNT signaling pathway from NCs (4.2%) to IBD colitis (39.7%) to IBD-associated neoplasia (63.4%) was seen (NCs vs. IBD colitis, p<0.01; IBD colitis vs. IBD-associated neoplasia, p=0.01). In the univariate logistic regression model, methylation of APC2 (OR 4.7, 95% CI: 1.1–20.63, p=0.04), SFRP1 (OR 5.1, 95% CI: 1.1–31.9, p=0.04), and SFRP2 (OR 5.1, 95% CI: 1.1–32.3, p=0.04) was associated with progression from IBD colitis to IBD-associated neoplasia, while APC1A methylation was borderline significant (OR 4.1, 95% CI: 0.95–17.5, p=0.06). In the multivariate logistic regression model, methylation of APC1A and APC2 was more likely to be associated with IBD-associated neoplasia than IBD colitis. (OR APC1A: 6.4, 95% CI: 1.1–37.7 p=0.04; OR APC2 9.1, 95% CI: 1.3–61.7, p=0.02).
Methylation of the WNT signaling genes is an early event seen in patients with IBD colitis and there is a progressive increase in methylation of the WNT signaling genes during development of IBD-associated neoplasia. Moreover, methylation of APC1A, APC2, SFRP1, and SFRP2 appears to mark progression from IBD colitis to IBD-associated neoplasia, and these genes may serve as biomarkers for IBD-associated neoplasia.
IBD; Methylation; WNT signaling; Colorectal cancer
Epigenetic therapy is emerging as a potential therapy for solid tumors. To investigate its mechanism of action, we performed integrative expression and methylation analysis of 63 cancer cell lines (breast, colorectal, and ovarian) after treatment with the DNA methyltransferase inhibitor 5-azacitidine (AZA). Gene Set Enrichment Analysis demonstrated significant enrichment for immunomodulatory pathways in all three cancers (14.4-31.3%) including interferon signaling, antigen processing and presentation, and cytokines/chemokines. Strong upregulation of cancer testis antigens was also observed. An AZA IMmune gene set (AIMs) derived from the union of these immunomodulatory pathway genes classified primary tumors from all three types into “high” and “low” AIM gene expression subsets in tumor expression data from both TCGA and GEO. Samples from selected patient biopsies showed upregulation of AIM genes after treatment with epigenetic therapy. These results point to a broad immune stimulatory role for DNA demethylating drugs in multiple cancers.
Epigenetics; immune; cancers; DNA methyltransferase inhibitor; interferon; methylation; antigen processing
Aberrant gene function and altered patterns of gene expression are key features of cancer. Growing evidence shows that acquired epigenetic abnormalities participate with genetic alterations to cause this dysregulation. Here, we review recent advances in understanding how epigenetic alterations participate in the earliest stages of neoplasia, including stem/precursor cell contributions, and discuss the growing implications of these advances for strategies to control cancer.
The importance of genetic and epigenetic alterations maybe in their aggregate role in altering core pathways in tumorigenesis.
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.
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. 6 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).
CRC that have silenced 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.
DNA Methylation; Extracellular Matrix Pathway; Prognostic Biomarker; Colorectal cancer
The ability to induce pluripotent stem cells from committed, somatic, human cells provides tremendous potential for regenerative medicine. However, there is a defined neoplastic potential inherent to such reprogramming that must be understood and may provide a model for understanding key events in tumorigenesis. Using genome wide assays we identify cancer-related epigenetic abnormalities that arise early during reprogramming and persist in induced pluripotent stem cell (iPS) clones. These include hundreds of abnormal gene silencing events, patterns of aberrant responses to epigenetic modifying drugs resembling those for cancer cells, and presence in iPS and partially reprogrammed cells of cancer-specific, gene promoter, DNA methylation alterations. Our findings suggest that by studying the process of induced reprogramming we may gain significant insight into the origins of epigenetic gene silencing associated with human tumorigenesis and add to means of assessing iPS for safety.
reprogramming; induced pluripotent stem cells (iPS); embryonic stem cells (ESC); DNA methylation; chromatin; cancer
Accumulating evidence points to a major role for chronic stress of cell renewal systems in the pathogenesis of important human diseases, including cancer, atherosclerosis and diabetes. Here we discuss emerging evidence that epigenetic abnormalities may make substantial contributions to these stress-induced pathologies. Although the mechanisms remain to be fully elucidated, we suggest that chronic stress can elicit heritable changes in the chromatin landscape that ‘lock’ cells in abnormal states, which then lead to disease. We emphasize the need to investigate epigenetic states in disease and links to stress and to consider how the knowledge gained through these studies may foster new means of disease prevention and management.
Somatic mutations in Isocitrate Dehydrogenase 1 (IDH1) are frequent in low grade and progressive gliomas and are characterized by the production of 2-hydroxyglutarate (2-HG) from α-ketoglutarate by the mutant enzyme. 2-HG is an “oncometabolite” that competitively inhibits α-KG dependent dioxygenases resulting in various widespread cellular changes including abnormal hypermethylation of genomic DNA and suppression of cellular differentiation. Despite the growing understanding of IDH mutant gliomas, the development of effective therapies has proved challenging in part due to the scarcity of endogenous mutant in vivo models. Here we report the generation of an endogenous IDH1 anaplastic astrocytoma model which rapidly grows in vivo, produces 2-HG and exhibits DNA hypermethylation. Using this model, we have demonstrated the preclinical efficacy and mechanism of action of the FDA approved demethylating drug 5-azacytidine in vivo. Long term administration of 5-azacytidine resulted in reduction of DNA methylation of promoter loci, induction of glial differentiation, reduction of cell proliferation and a significant reduction in tumor growth. Tumor regression was observed at 14 weeks and subsequently showed no signs of re-growth at 7 weeks despite discontinuation of therapy. These results have implications for clinical trials of demethylating agents for patients with IDH mutated gliomas.
IDH; 5-azacytidine; progressive glioma; xenograft; astrocytoma; methylation
Little information is available on genetic and epigenetic changes in duodenal adenocarcinomas. The purpose was to identify possible subsets of duodenal adenocarcinomas based on microsatellite instability (MSI), DNA methylation, mutations in the KRAS and BRAF genes, clinicopathologic features, and prognosis.
Demographics, tumor characteristics and survival were available for 99 duodenal adenocarcinoma patients. Testing for KRAS and BRAF mutations, MSI, MLH1 methylation and CpG island methylator phenotype (CIMP) status was performed. A Cox proportional hazard model was built to predict survival.
CIMP+ was detected in 27 of 99 (27.3%) duodenal adenocarcinomas, and was associated with MSI (P = 0.011) and MLH1 methylation (P < 0.001), but not with KRAS mutations (P = 0.114), as compared to CIMP− tumors. No BRAF V600E mutation was detected. Among the CIMP+ tumors, 15 (55.6%) were CIMP+/MLH1-unmethylated (MLH1-U). Kaplan-Meier analysis showed tumors classified by CIMP, CIMP/MLH1 methylation status or CIMP/MSI status could predict overall survival (OS; P = 0.047, 0.002, and 0.002, respectively), while CIMP/MLH1 methylation status could also predict time-to-recurrence (TTR; P = 0.016). In multivariate analysis, CIMP/MLH1 methylation status showed a significant prognostic value regarding both OS (P < 0.001) and TTR (P = 0.023). Patients with CIMP+/MLH1-U tumors had the worst OS and TTR.
Our results demonstrate existence of CIMP in duodenal adenocarcinomas. The combination of CIMP+/MLH1-U appears to be independently associated with poor prognosis in patients with duodenal adenocarcinomas. This study also suggests that BRAF mutations are not involved in duodenal tumorigenesis, MSI or CIMP development.
Duodenal Adenocarcinoma; CpG Island Methylator Phenotype; Microsatellite Instability; Prognosis
The tumor suppressor gene HYPERMETHYLATED IN CANCER 1 (HIC1), which encodes a transcriptional repressor, is epigenetically inactivated in various human cancers. Here, we show that HIC1 is a direct transcriptional repressor of the gene encoding ephrin-A1, a cell surface ligand implicated in the pathogenesis of epithelial cancers. We also show that mouse embryos lacking both Hic1 alleles manifest developmental defects spatially associated with misexpression of ephrin-A1, and that overexpression of ephrin-A1 is a feature of tumors arising in Hic1 heterozygous mice in which the remaining wild-type allele is epigenetically silenced. In breast cancer, we find that ephrin-A1 expression is common in vivo, but that in cell culture, expression of the EphA receptors is predominant. Restoration of HIC1 function in breast cancer cells leads to a reduction in tumor growth in vivo, an effect that can be partially rescued by co-overexpression of ephrin-A1. Interestingly, overexpression of ephrin-A1 in vitro triggers downregulation of EphA2 and EphA4 levels, resulting in an expression pattern similar to that seen in vivo. We conclude that Hic1 spatially restricts ephrin-A1 expression in development, and that upregulated expression of ephrin-A1 resulting from epigenetic silencing of HIC1 in cancer cells may be an important mechanism in epithelial malignancy.
Hic1; Ephrin-A1; EphA2; EphA4
Recent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke exposure.
We show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44hi/CD24low population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis.
Our results indicate that exposure to cigarette smoke leads to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state.
Tobacco; Breast cancer; Cell motility and invasion; Epithelial to mesenchymal transition; Metastasis; Intraductal injection
The promise of targeting epigenetic abnormalities for cancer therapy has not been realized for solid tumours, although increasing evidence is demonstrating its worth in haematological malignancies. In fact, true clinical efficacy in haematopoietic-related neoplasms has only become evident at low doses of epigenetic-targeting drugs (namely, inhibitors of histone deacetylase and DNA methyltransferases). Describing data from preclinical studies and early clinical trial results, we hypothesize that in using low-dose epigenetic-modulating agents, tumour cells can be reprogrammed, which overrides any immediate cytotoxic and off-target effect observed at high dose. We suggest that such optimization of drug dosing and scheduling of currently available agents could give these agents a prominent place in cancer management—when used alone or in combination with other therapies. If so, optimal use of these known agents might also pave the way for the introduction of other agents that target the epigenome.
Downregulation of the tight junction protein claudin 1 is a frequent event in breast cancer and is associated with recurrence, metastasis, and reduced survival, suggesting a tumor suppressor role for this protein. Tumor suppressor genes are often epigenetically silenced in cancer. Downregulation of claudin 1 via DNA promoter methylation may thus be an important determinant in breast cancer development and progression. To investigate if silencing of claudin 1 has an epigenetic etiology in breast cancer we compared gene expression and methylation data from 217 breast cancer samples and 40 matched normal samples available through the Cancer Genome Atlas (TCGA). Moreover, we analyzed claudin 1 expression and methylation in 26 breast cancer cell lines. We found that methylation of the claudin 1 promoter CpG island is relatively frequent in estrogen receptor positive (ER+) breast cancer and is associated with low claudin 1 expression. In contrast, the claudin 1 promoter was not methylated in most of the ER-breast cancers samples and some of these tumors overexpress claudin 1. In addition, we observed that the demethylating agents, azacitidine and decitabine can upregulate claudin 1 expression in breast cancer cell lines that have a methylated claudin 1 promoter. Taken together, our results indicate that DNA promoter methylation is causally associated with downregulation of claudin 1 in a subgroup of breast cancer that includes mostly ER+ tumors, and suggest that epigenetic therapy to restore claudin 1 expression might represent a viable therapeutic strategy in this subtype of breast cancer.
To evaluate the methylation state of 31 genes in sputum as biomarkers in an expanded nested, case-control study from the Colorado Cohort and to assess the replication of results from the most promising genes in an independent case-control study of asymptomatic Stage I lung cancer patients from New Mexico.
Cases and controls from Colorado and New Mexico were interrogated for methylation of up to 31 genes using nested, methylation specific PCR. Individual genes and methylation indices were used to assess the association between methylation and lung cancer with logistic regression modeling.
Seventeen genes with odds ratios of 1.4 – 3.6 were identified and selected for replication in the New Mexico study. Overall, the direction of effects seen in New Mexico was similar to Colorado with the largest increase in case discrimination (odds ratios, 3.2 – 4.2) seen for the PAX5α, GATA5, and SULF2 genes. ROC curves generated from seven gene panels from Colorado and New Mexico studies showed prediction accuracy of 71% and 77%, respectively. A 22-fold increase in lung cancer risk was seen for a subset of New Mexico cases with five or more genes methylated. Sequence variants associated with lung cancer did not improve the accuracy of this gene methylation panel.
These studies have identified and replicated a panel of methylated genes whose integration with other promising biomarkers could initially identify the highest risk smokers for computed tomography screening for early detection of lung cancer.
gene methylation; sputum; lung cancer; biomarker
Promoter hypermethylation is a prevalent phenomenon, found in virtually all cancer types studied thus far, and accounts for tumor suppressor gene silencing in the absence of genetic mutations. The mechanism behind the establishment and maintenance of such aberrant hypermethylation has been under intense study. Here, we have uncovered a link between aberrant gene silencing associated with promoter CpG island DNA methylation and the siRNA/miRNA processing enzyme, DICER, in human cancer cells. By comparing demethylated HCT116 colon cancer cells with HCT116 cells genetically rendered hypomorphic for DICER, we identified a group of epigenetically silenced genes that became reactivated in the absence of functional DICER. This reactivation is associated with a dramatic loss of localized promoter DNA hypermethylation. Thus, intact DICER is required to maintain full promoter DNA hypermethylation of select epigenetically silenced loci in human cancer cells.
SRY-box containing gene 17 (Sox17) is a member of the high mobility group (HMG) transcription factor superfamily, which plays critical roles in the regulation of development and stem/precursor cell function, at least partly through repression of Wnt pathway activity. Modulators controlling aberrant Wnt signaling activation are frequently disrupted in human cancers through complementary effects of epigenetic and genetic changes. Our recent global analysis of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that SOX17 gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. Here, we report that CpG island methylation-dependent silencing of SOX17 occurs in 100% of CRC cell lines, 86% of colorectal adenomas, 100% of stage I and II CRC, 89% of stage III CRC, 89% of primary esophageal cancer, and 50% of non–small cell lung cancer. Overexpression of SOX17 in HCT116 CRC cells inhibits colony growth and β-catenin/T-cell factor–dependent transcription. Structure-based deletion analysis further shows the presence of a Wnt signaling repression domain in the SOX17 HMG box. Together, our studies suggest that SOX17 is a negative modulator of canonical Wnt signaling, and that SOX17 silencing due to promoter hypermethylation is an early event during tumorigenesis and may contribute to aberrant activation of Wnt signaling in CRC.
Epigenetic gene regulation is a key determinant of heritable gene expression patterns and is critical for normal cellular function. Dysregulation of epigenetic transcriptional control is a fundamental feature of cancer, particularly manifesting as increased promoter DNA methylation with associated aberrant gene silencing which plays a significant role in tumor progression. We now globally map key chromatin parameters for genes with promoter CpG island DNA hypermethylation in colon cancer cells by combining micraoarray gene expression analyses with ChIP on chip technology. We first show that the silent state of such genes universally correlates with a broad, low level distribution of the PcG mediated histone modification, methylation of lysine 27 of histone 3 (H3K27me) and a very low level of the active mark, H3K4me2. This chromatin pattern, and particularly H3K4me2 levels, crisply separates DNA hypermethylated genes from those where histone deacetylation is responsible for transcriptional silencing. Moreover, the chromatin pattern can markedly enhance identification of truly silent and DNA hypermethylated genes. We additionally find that when DNA hypermethylated genes are de-methylated and re-expressed, they adopt a “bivalent” chromatin pattern which is associated with the poised gene expression state of a large group of ES cell genes, and is characterized by an increase in levels of both the H3K27me3 and H3K4me2 marks. Our data have great relevance for the increasing interest in re-expression of DNA hypermethylated genes for the treatment of cancer.
DNA methylation; chromatin; histone modifications; cancer; epigenetic