A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing.

Adult cancers may derive from stem or early progenitor cells. Epigenetic modulation of gene expression is essential for normal function of these early cells but is highly abnormal in cancers, which often show aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors. We find that for such genes, both normal and malignant embryonic cells generally lack the hypermethylation of DNA found in adult cancers. In embryonic stem cells, these genes are held in a 'transcription-ready' state mediated by a 'bivalent' promoter chromatin pattern consisting of the repressive mark, histone H3 methylated at Lys27 (H3K27) by Polycomb group proteins, plus the active mark, methylated H3K4. However, embryonic carcinoma cells add two key repressive marks, dimethylated H3K9 and trimethylated H3K9, both associated with DNA hypermethylation in adult cancers. We hypothesize that cell chromatin patterns and transient silencing of these important regulatory genes in stem or progenitor cells may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing during tumor initiation and progression.

Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation.

Double-stranded RNA molecules targeted to gene promoter regions can induce transcriptional gene silencing in a DNA cytosine methylation-dependent manner in plants (RNA-dependent DNA methylation). Whether a similar mechanism exists in mammalian systems is a vital and controversial issue. DNA methylation is an important component in mammalian gene silencing for normal processes such as gene imprinting and X-chromosome inactivation, and aberrant CpG island hypermethylation at tumor-suppressor promoters is associated with transcriptional silencing and loss of gene function in cancer. Hence, we investigated whether RNA-dependent DNA methylation might operate in human cancers to mediate epigenetic silencing using the endogenous gene CDH1 as a potential target. The loss of this cell-cell adhesion factor facilitates the metastatic process, and its promoter is frequently hypermethylated in breast and other cancers. We found that, although small double-stranded RNAs targeted exclusively to the CDH1 promoter could effectively induce transcriptional repression with chromatin changes characteristic of inactive promoters, this was entirely independent of DNA methylation. Moreover, we could accomplish such silencing in a cancer cell line genetically modified to lack virtually any capacity to methylate DNA.

CpG island hypermethylation is maintained in human colorectal cancer cells after RNAi-mediated depletion of DNMT1.

The role of the primary mammalian DNA methyltransferase, DNMT1, in maintaining CpG island methylation in human colon cancer cells has recently been questioned. This controversy has arisen from discrepancies between genetic knockout and RNA interference-mediated knockdown studies. Here, we re-examined the RNA interference-based approach and found that hypermethylation of single-copy genes is maintained in cells transiently and stably depleted of DNMT1.

Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer.

Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding beta-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free beta-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.

Prognostic significance of Gremlin1 (GREM1) promoter CpG island hypermethylation in clear cell renal cell carcinoma.

Gremlin1 (GREM1), a bone morphogenetic protein antagonist and putative angiogenesis-modulating gene, is silenced by promoter hypermethylation in human malignancies. Here we study GREM1 methylation in clear cell renal cell carcinoma (ccRCC) and its impact on tumor characteristics and clinical outcome. Three GREM1 promoter CpG island regions (i, ii, iii) were analyzed by methylation-specific PCR and/or bisulfite sequencing in ccRCC cell lines and ccRCCs from two independent patient series. Results were correlated with clinicopathological and angiogenic parameters. Bisulfite sequencing of ccRCC cell lines showed GREM1 methylation, associated with absence of GREM1 mRNA. GREM1 methylation prevalence in ccRCCs varied between regions: 55%, 24%, and 20% for regions i, ii, and iii, respectively. GREM1 region iii methylation was associated with increased tumor size (P = 0.02), stage (P = 0.013), grade (P = 0.04), tumor (P = 0.001), and endothelial cell (P = 0.0001) proliferation and decreased mean vessel density (P = 0.001) in a hospital-based ccRCC series (n = 150). In univariate analysis, GREM1 region iii methylated ccRCCs had a significant worse survival when compared with unmethylated ccRCCs (hazard ratio [HR] = 2.35, 95% confidence interval [CI]:1.29 to 4.28), but not in multivariate analysis (HR = 0.88, 95% CI: 0.45 to 1.74). In a population-based validation series (n = 185), GREM1 region iii methylation was associated with increased Fuhrman grade (P = 0.03) and decreased overall survival (P = 0.001) in univariate and multivariate analysis (HR = 2.32, 95% CI: 1.52 to 3.53 and HR = 2.27, 95% CI: 1.44 to 3.59, respectively). The strong correlation between GREM1 region iii promoter methylation and increased malignancy and its correlation with active angiogenesis indicates a role for GREM1 in ccRCC carcinogenesis and tumor angiogenesis.

Comparing the DNA hypermethylome with gene mutations in human colorectal cancer.

We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumor-specific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken.

Concomitant promoter methylation of multiple genes in lung adenocarcinomas from current, former and never smokers.

Aberrant promoter hypermethylation is one of the major mechanisms in carcinogenesis and some critical growth regulatory genes have shown commonality in methylation across solid tumors. Twenty-six genes, 14 identified through methylation in colon and breast cancers, were evaluated using primary lung adenocarcinomas (n = 175) from current, former and never smokers. Tumor specificity of methylation was validated through comparison of 14 lung cancer cell lines to normal human bronchial epithelial cells derived from bronchoscopy of 20 cancer-free smokers. Twenty-five genes were methylated in 11-81% of primary tumors. Prevalence for methylation of TNFRSF10C, BHLHB5 and BOLL was significantly higher in adenocarcinomas from never smokers than smokers. The relation between methylation of individual genes was examined using pairwise comparisons. A significant association was seen between 138 (42%) of the possible 325 pairwise comparisons. Most notably, methylation of MMP2, BHLHB4 or p16 was significantly associated with methylation of 16-19 other genes, thus predicting for a widespread methylation phenotype. Kaplan-Meier log-rank test and proportional hazard models identified a significant association between methylation of SULF2 (a pro-growth, -angiogenesis and -migration gene) and better patient survival (hazard ratio = 0.23). These results demonstrate a high degree of commonality for targeted silencing of genes between lung and other solid tumors and suggest that promoter hypermethylation in cancer is a highly co-ordinated event.

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.

Convergence of mutation and epigenetic alterations identifies common genes in cancer that predict for poor prognosis.

BACKGROUND: The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities. Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer. However, determining which of these many genes play key roles in cancer development has proven challenging. Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific. We hypothesize that key tumor suppressor genes in cancer may be subject to mutation or hypermethylation. METHODS AND FINDINGS: Here, we show that combined genetic and epigenetic analysis of these genes reveals many with a higher putative tumor suppressor status than would otherwise be appreciated. At least 36 of the 189 genes newly recognized to be mutated are targets of promoter CpG island hypermethylation, often in both colon and breast cancer cell lines. Analyses of primary tumors show that 18 of these genes are hypermethylated strictly in primary cancers and often with an incidence that is much higher than for the mutations and which is not restricted to a single tumor-type. In the identical breast cancer cell lines in which the mutations were identified, hypermethylation is usually, but not always, mutually exclusive from genetic changes for a given tumor, and there is a high incidence of concomitant loss of expression. Sixteen out of 18 (89%) of these genes map to loci deleted in human cancers. Lastly, and most importantly, the reduced expression of a subset of these genes strongly correlates with poor clinical outcome. CONCLUSIONS: Using an unbiased genome-wide approach, our analysis has enabled the discovery of a number of clinically significant genes targeted by multiple modes of inactivation in breast and colon cancer. Importantly, we demonstrate that a subset of these genes predict strongly for poor clinical outcome. Our data define a set of genes that are targeted by both genetic and epigenetic events, predict for clinical prognosis, and are likely fundamentally important for cancer initiation or progression.

Differential requirement for DNA methyltransferase 1 in maintaining human cancer cell gene promoter hypermethylation.

Previous work has shown that DNA hypermethylation of tumor suppressor genes in colorectal cancer cells may be maintained in the absence of the major mammalian methyltransferase, DNA methyltransferase 1 (DNMT1). In an effort to dissect the dependency on DNMT1 to maintain such hypermethylation in different cancer types, we performed a systematic analysis of depletion of DNMT1 in colorectal (SW48), bladder (T24), and breast (T47D) cancer cells by DNMT1-specific small hairpin RNA (shRNA) targeting. We show that although DNMT1-deficient SW48 and T24 cells exhibited no observable growth defects and were able to maintain promoter hypermethylation, DNMT1-deficient T47D breast cells failed to form comparable numbers of colonies when stably selected for the incorporation of the DNMT1-specific shRNA expression vector, suggesting a growth defect with reduced levels of DNMT1. Further treatment of T47D cells with transient transfection of small interfering RNA targeting DNMT1 revealed that severely DNMT1-deficient T47D cells could not fully maintain promoter hypermethylation, and gene silencing was partially reversed at two of the three assayed loci. These observations suggest that human cancer cells may differ in their reliance on DNMT1 for maintaining DNA methylation.

De novo CpG island methylation in human cancer cells.

A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT 3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells.

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.

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.

Regulation of neuroendocrine differentiation in gastrointestinal carcinoid tumor cells by notch signaling.

CONTEXT: Gastrointestinal (GI) carcinoid tumors elaborate serotonin and other vasoactive substances, causing the carcinoid syndrome. Based on developmental biology data, we hypothesized that basic helix-loop-helix transcription factors, including achaete-scute complex homolog-like 1 (Ascl1)/hASH1, and the Notch signaling pathway might regulate the neuroendocrine phenotype in GI carcinoids. OBJECTIVE: The aim of this study was to evaluate expression of developmental transcription factors and Notch signaling components in GI carcinoids and model their interaction in a relevant GI carcinoid cell line. DESIGN: Fourteen GI carcinoid tumor specimens, five paired adjacent normal tissues, fetal tissues, and tumor cell lines were analyzed by RT-PCR and immunoblot. BON carcinoid cells were further analyzed after Notch overexpression for neuroendocrine marker expression, serotonin production, and growth. SETTING: The study was conducted in an academic referral center. PATIENTS OR OTHER PARTICIPANTS: Deidentified archival pathology specimens were examined. RESULTS: Among a panel of six developmental transcription factors tested, only Ascl1 mRNA was overexpressed compared with surrounding normal tissue (seven of 10 GI carcinoid tumors and in BON cells, none of five normal tissues). Ascl1 protein was also expressed in four of four carcinoid tumors and BON cells). Notch pathway ligands, receptors, and downstream effectors were widely expressed in tumor and normal specimens. Overexpression of activated Notch1 in BON cells led to induction of the Notch effector hairy and enhancer of split 1 (Hes1), loss of Ascl1, reductions in neuron-specific enolase, synaptophysin, and chromogranin A, and most significantly, an 89% decrease in serotonin concentration and equivalent reductions in serotonin-reactive cells and repression of tryptophan hydroxylase 1 mRNA. CONCLUSIONS: The Notch signaling pathway is a significant regulator of neuroendocrine differentiation and serotonin production in GI carcinoid tumors.

Epigenetic silencing of neurofilament genes promotes an aggressive phenotype in breast cancer.

Neurofilament heavy polypeptide (NEFH) has recently been identified as a candidate DNA hypermethylated gene within the functional breast cancer hypermethylome. NEFH exists in a complex with neurofilament medium polypeptide (NEFM) and neurofilament light polypeptide (NEFL) to form neurofilaments, which are structural components of the cytoskeleton in mature neurons. Recent studies reported the deregulation of these proteins in several malignancies, suggesting that neurofilaments may have a role in other cell types as well. Using a comprehensive approach, we studied the epigenetic inactivation of neurofilament genes in breast cancer and the functional significance of this event. We report that DNA methylation-associated silencing of NEFH, NEFL, and NEFM in breast cancer is frequent, cancer-specific, and correlates with clinical features of disease progression. DNA methylation-mediated inactivation of these genes occurs also in multiple other cancer histologies including pancreas, gastric, and colon. Restoration of NEFH function, the major subunit of the neurofilament complex, reduces proliferation and growth of breast cancer cells and arrests them in Go/G1 phase of the cell cycle along with a reduction in migration and invasion. These findings suggest that DNA methylation-mediated silencing of the neurofilament genes NEFH, NEFM, and NEFL are frequent events that may contribute to the progression of breast cancer and possibly other malignancies.

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.

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.

Frequent inactivation of cysteine dioxygenase type 1 contributes to survival of breast cancer cells and resistance to anthracyclines.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

Biomarkers for detection and prognosis of breast cancer identified by a functional hypermethylome screen.

Breast cancer (BC) is a disease with diverse tumor heterogeneity, which challenges conventional approaches to develop biomarkers for early detection and prognosis. To identify effective biomarkers, we performed a genome-wide screen for functional methylation changes in BC, i.e., genes silenced by promoter hypermethylation, using a functionally proven gene expression approach. A subset of candidate hypermethylated genes were validated in primary BCs and tested as markers for detection and prognosis prediction of BC. We identified 33 cancer specific methylated genes and, among these, two categories of genes: (1) highly frequent methylated genes that detect early stages of BC. Within that category, we have identified the combination of NDRG2 and HOXD1 as the most sensitive (94%) and specific (90%) gene combination for detection of BC; (2) genes that show stage dependent methylation frequency pattern, which are candidates to help delineate BC prognostic signatures. For this category, we found that methylation of CDO1, CKM, CRIP1, KL and TAC1 correlated with clinical prognostic variables and was a significant prognosticator for poor overall survival in BC patients. CKM [Hazard ratio (HR) = 2.68] and TAC1 (HR = 7.73) were the strongest single markers and the combination of both (TAC1 and CKM) was associated with poor overall survival independent of age and stage in our training (HR = 1.92) and validation cohort (HR = 2.87). Our study demonstrates an efficient method to utilize functional methylation changes in BC for the development of effective biomarkers for detection and prognosis prediction of BC.