Publications

Background Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required. Methodology/Principal Findings We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients. Conclusions/Significance Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.

Abstract miR-9 was initially identified as an epigenetically regulated miRNA in tumours, but inconsistent findings have been reported so far. We analysed the expression of miR-9-5p, miR-9-3p, pri-miRs and MIR9 promoters methylation status in 131 breast cancer cases and 12 normal breast tissues (NBTs). The expression of both mature miRs was increased in tumours as compared to NBTs (P < 0.001) and negatively correlated with ER protein expression ( P = 0.005 and P = 0.003, for miR-9-3p and miR-9-5p respectively). In addition, miR-9-5p showed a significant negative correlation with PgR (P = 0.002). Consistently, miR-9-5p and miR-9 3p were differentially expressed in the breast cancer subgroups identified by ER and PgR expression and HER2 amplification. No significant correlation between promoter methylation and pri-miRNAs expressions was found either in tumours or in NBTs. In the Luminal breast cancer subtype the expression of miR-9-5p was associated with a worse prognosis in both univariable and multivariable analyses. Ingenuity Pathway Analysis exploring the putative interactions among miR-9-5p/miR-9-3p, ER and PgR upstream and downstream regulators suggested a regulatory loop by which miR-9-5p but not miR-9-3p is induced by steroid hormone receptor and acts within hormone-receptor regulated pathways.

BRG1, also called SMARCA4, is the catalytic subunit of the SWI/SNF chromatin-remodelling complex and influences transcriptional regulation by disrupting histone-DNA contacts in an ATP-dependent manner. BRG1 and other members of the SWI/SNF complex become inactivated in tumours, implying a role in cancer development. To understand the contribution of BRG1 to lung tumourigenesis, we restored BRG1 in H1299 lung cancer cells and used cDNA microarray analysis to identify changes in gene expression. Forty-three transcripts became activated, whereas two were repressed. Chromatin immunoprecipitation of resulting candidate genes revealed that the CYP3A4 and ZNF185 promoters recruited BRG1 and that recruitment to the CYP3A4 promoter was specific to this gene and did not involve the CYP3A5 or CYP3A7 family members. Moreover, specifically BRG1 but not its homologue BRM was recruited to the CYP3A4 and ZNF185 promoters. To explore their potential relevance in lung tumours, levels of CYP3A4 and ZNF185 transcripts were evaluated in seven additional lung cancer cell lines. CYP3A4 was undetectable in any of the lung cancer cells tested, and only the CYP3A5 family member was present in the A549 and Calu-3 cells. In contrast, the amount of ZNF185 transcript clearly varied among lung cancer cell lines and severely reduced levels were observed in BRG1-deficient cells, except those of A427. We extended these observations to 27 lung primary tumours using real-time RT-PCR (TaqMan) and observed that four (15%) and 14 (52%) of them had BRG1 and ZNF185 downregulation, respectively, when compared with normal lung. No significant correlation between reduced levels of BRG1 and ZNF185 was observed, indicating that additional mechanisms to BRG1 inactivation may contribute to the loss of ZNF185 expression in lung tumours. In conclusion, our results provide evidence that transcriptional activation of ZNF185 and CYP3A4 is mediated by direct association of BRG1 with their promoters and also indicate that a decreased level of ZNF185 is a common feature of lung tumours and may be of biological relevance in lung carcinogenesis.

<div>Abstract<p>Differentially methylated oral squamous cell carcinoma (OSCC) biomarkers, identified <i>in vitro</i> and validated in well-characterized surgical specimens, have shown poor clinical correlation in cohorts with different risk profiles.</p><p>To overcome this lack of relevance, we used the HumanMethylation27 BeadChip, publicly available methylation and expression array data, and quantitative methylation specific PCR to uncover differential methylation in OSCC clinical samples with heterogeneous risk profiles.</p><p>A two stage design consisting of discovery and prevalence screens was used to identify differential promoter methylation and deregulated pathways in patients diagnosed with OSCC and head and neck squamous cell carcinoma.</p><p>Promoter methylation of <i>KIF1A</i> (κ = 0.64), <i>HOXA9</i> (κ = 0.60), <i>NID2</i> (κ = 0.60), and <i>EDNRB</i> (κ = 0.60) had a moderate to substantial agreement with clinical diagnosis in the discovery screen. <i>HOXA9</i> had 68% sensitivity, 100% specificity, and a 0.81 Area Under the Curve (AUC). <i>NID2</i> had 71% sensitivity, 100% specificity, and a 0.79 AUC. In the prevalence screen, <i>HOXA9</i> (κ = 0.82) and <i>NID2</i> (κ = 0.80) had an almost perfect agreement with histologic diagnosis. <i>HOXA9</i> had 85% sensitivity, 97% specificity, and a 0.95 AUC. <i>NID2</i> had 87% sensitivity, 95% specificity, and a 0.91 AUC. A <i>HOXA9</i> and <i>NID2</i> gene panel had 94% sensitivity, 97% specificity, and a 0.97 AUC. In saliva, from OSCC cases and controls, <i>HOXA9</i> had 75% sensitivity, 53% specificity, and a 0.75 AUC. <i>NID2</i> had 87% sensitivity, 21% specificity, and a 0.73 AUC.</p><p>This phase I Biomarker Development Trial identified a panel of differentially methylated genes in normal and OSCC clinical samples from patients with heterogeneous risk profiles. This panel may be useful for early detection and cancer prevention studies. <i>Cancer Prev Res; 4(7); 1061–72. ©2011 AACR</i>.</p></div>

<p>Figure legends for Supplementary Figures</p>

Clinico-pathological features of the second CRC patient cohort. (XLSX 10 kb)

Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients.We performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing.Epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients.Our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients.

Supplementary Table 2 from Discovery of Epigenetically Silenced Genes by Methylated DNA Immunoprecipitation in Colon Cancer Cells

<p>Clinicopathologic information of the studied MDS patients</p>

PLoS Genetics

Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these 2 exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at <i>CYP2E1</i>(β_Non-exposed = 0.06, β_SRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of <i>CYP2E1</i> regulatory regions in an expanded cohort, higher DNA methylation status—both the mean across 16 CpG sites (<i>P</i> &lt; 0.01) and at each specific CpG site (<i>P</i> &lt; 0.05)—was associated with exposure to lower 3rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood x SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (<i>P</i> = 0.04), CpG9 (<i>P</i> = 0.04) and CpG10 (<i>P</i> = 0.02), in the interrogated <i>CYP2E1</i> region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal <i>CYP2E1</i> DNA methylation status, which, in turn, appeared to be associated with birth weight.