Publications

Cancer is as much an epigenetic disease as a genetic one; however, the interplay between these two processes is unclear. Recently, it has been shown that a large proportion of DNA methylation variability can be explained by allele-specific methylation (ASM), either at classical imprinted loci or those regulated by underlying genetic variants. During a recent screen for imprinted differentially methylated regions, we identified the genomic interval overlapping the non-coding nc886 RNA (previously known as vtRNA2-1) as an atypical ASM that shows variable levels of methylation, predominantly on the maternal allele in many tissues. Here we show that the nc886 interval is the first example of a polymorphic imprinted DMR in humans. Further analysis of the region suggests that the interval subjected to ASM is approximately 2 kb in size and somatically acquired. An in depth analysis of this region in primary cancer samples with matching normal adjacent tissue from the Cancer Genome Atlas revealed that aberrant methylation in bladder, breast, colon and lung tumors occurred in approximately 27% of cases. Hypermethylation occurred more frequently than hypomethylation. Using additional normal-tumor paired samples we show that on rare occasions the aberrant methylation profile is due to loss-of-heterozygosity. This work therefore suggests that the nc886 locus is subject to variable allelic methylation that undergoes cancer-associated epigenetic changes in solid tumors.

Abstract Background: Previous studies support a tumor-suppressor role for LRRC3B across various types of cancers. We aimed to investigate the association between DNA methylation of LRRC3B and overall survival (OS) for patients with early-stage non–small cell lung cancer (NSCLC). Methods: This study included 1,230 patients with early-stage NSCLC. DNA was extracted from lung tumor tissues and DNA methylation was measured using Illumina Infinium HumanMethylation450 BeadChips. The association between DNA methylation and OS was first tested using Cox regression on a discovery cohort and then validated in an independent cohort. Next, the association between DNA methylation and gene expression was investigated in two independent cohorts. Finally, the association between gene expression and OS was investigated in three independent groups of patients. Results: Three novel DNA methylation sites in LRRC3B were significantly associated with OS in two groups of patients. Patients with hypermethylation in the DNA methylation sites had significantly longer survival than the others in both the discovery cohort (HR, 0.62; P = 2.02 × 10−05) and validation cohort (HR, 0.55; P = 4.44 × 10−04). The three DNA methylation sites were significantly associated with LRRC3B expression, which was also associated with OS. Conclusions: Using clinical data from a large population, we illustrated the association between DNA methylation of LRRC3B and OS of early-stage NSCLC. Impact: We provide evidence of plausibility for building biomarkers on DNA methylation of LRRC3B for OS of early-stage NSCLC, thus filling a gap between previous in vitro studies and clinical applications.

<p>PDF file - 574K, Microarray pre-processing.</p>

Abstract Background Current noninvasive assays have limitations in the early detection of colorectal cancer. We evaluated the clinical utility of promoter methylation of the long noncoding RNA LINC00473 as a noninvasive biomarker to detect colorectal cancer and associated precancerous lesions. Methods We evaluated the epigenetic regulation of LINC00473 through promoter hypermethylation in colorectal cancer cell lines using bisulfite genomic sequencing and expression analyses. DNA methylation of LINC00473 was analyzed in primary colorectal tumors using 450K arrays and RNA-seq from The Cancer Genome Atlas (TCGA). Tissue-based findings were validated in several independent cohorts of colorectal cancer and advanced colorectal polyp patients by pyrosequencing. We explored the clinical utility of LINC00473 methylation for the early detection of colorectal cancer in plasma cell-free DNA by quantitative methylation-specific PCR and droplet digital PCR. Results LINC00473 showed transcriptionally silencing due to promoter hypermethylation in colorectal cancer cell lines and primary tumors. Methylation of the LINC00473 promoter accurately detected primary colorectal tumors in two independent clinical cohorts, with areas under the receiver operating characteristic curves (AUCs) of 0.94 and 0.89. This biomarker also identified advanced colorectal polyps from two other tissue-based clinical cohorts with high diagnostic accuracy (AUCs of 0.99 and 0.78). Finally, methylation analysis of the LINC00473 promoter in plasma cell-free DNA accurately identified patients with colorectal cancer and advanced colorectal polyps (AUCs of 0.88 and 0.84, respectively), which was confirmed in an independent cohort of patients. Conclusions Hypermethylation of the LINC00473 promoter is a new promising biomarker for noninvasive early detection of colorectal cancer and related precancerous lesions.

Abnormal fatty acid metabolism and availability are landmarks of metabolic diseases, which in turn are associated with aberrant DNA methylation profiles. To understand the role of fatty acids in disease epigenetics, we sought DNA methylation profiles specifically induced by arachidonic or oleic acid (AA and OA) in cultured cells and compared those with published profiles of normal and diseased tissues. THP‐1 monocytes were stimulated with AA or OA and analyzed using Infinium HumanMethylation450 BeadChip (Illumina) and Human Exon 1.0 ST array (Affymetrix). Data were corroborated in mouse embryonic fibroblasts. Comparisons with publicly available data were conducted by standard bioinformatics. AA and OA elicited a complex response marked by a general DNA hyper‐ and hypomethylation in the 1–200 μM range, respectively, with a maximal differential response at the 100 μM dose. The divergent response to AA and OA was prominent within the gene body of target genes, where it correlated positively with transcription. AA‐induced DNA methylation profiles were similar to the corresponding profiles described for palmitic acid, atherosclerosis, diabetes, obesity and autism, but relatively dissimilar from OA‐induced profiles. A set of genes was identified, that display gene body hypermethylation in response to AA and in human atherosclerosis. Biochemical evidence pointed to beta‐oxidation, PPAR‐alpha and sirtuin 1 as important mediators of AA‐induced DNA methylation changes. In conclusion, AA and OA exert distinct effects on the DNA methylome. The observation that AA may contribute to shape the epigenome of important metabolic diseases supports and expands current diet‐based therapeutic and preventive efforts. Support or Funding Information CONCyTEG grant no. 08‐03‐K662‐020‐A01, Mexican Council for Science and Technology (CONACyT) Basic Science (Ciencia Básica) grant no. 83401 to GL; CONACyT Sabbatical Fellowships no. 166209 to G.L. and no. 166058 to S.Z.; CONACyT Ph.D. Fellowship no. 334370 to G.A.S.‐M.; the Spanish Ministry of Ministry of Economy and Competitiveness (MINECO) grant SAF2011‐25619 to A.V.

<p>Resistant in parallel with parental clones of Karpas1718 cell line were exposed to a library of 348 compounds alone (single, 1µM) or in combination with 1µM idelalisib (combo) for 72h. Cell viability was measured by MTT assay (72h). Yellow for benefit in combo, purple benefit in single treatment.</p>

<div>Abstract<p>The development of resistance to tyrosine kinase inhibitors (TKI) limits the long-term efficacy of cancer treatments involving them. We aimed to understand the mechanisms that underlie acquired resistance (AR) to MET inhibitors in lung cancer. EBC1 cells, which have <i>MET</i> amplification and are sensitive to TKIs against MET, were used to generate multiple clones with AR to a MET-TKI. Whole-exome sequencing, RNA sequencing, and global DNA methylation analysis were used to scrutinize the genetic and molecular characteristics of the resistant cells. AR to the MET-TKI involved changes common to all resistant cells, that is, phenotypic modifications, specific changes in gene expression, and reactivation of AKT, ERK, and mTOR. The gene expression, global DNA methylation, and mutational profiles distinguished at least two groups of resistant cells. In one of these, the cells have acquired sensitivity to erlotinib, concomitantly with mutations of the <i>KIRREL, HDAC11, HIATL1</i>, and <i>MAPK1IP1L</i> genes, among others. In the other group, some cells have acquired inactivation of neurofibromatosis type 2 (<i>NF2</i>) concomitantly with strong overexpression of <i>NRG1</i> and a mutational profile that includes changes in <i>LMLN</i> and <i>TOMM34</i>. Multiple independent and simultaneous strategies lead to AR to the MET-TKIs in lung cancer cells. The acquired sensitivity to erlotinib supports the known crosstalk between MET and the HER family of receptors. For the first time, we show inactivation of <i>NF2</i> during acquisition of resistance to MET-TKI that may explain the refractoriness to erlotinib in these cells. <i>Mol Cancer Ther; 16(7); 1366–76. ©2017 AACR</i>.</p></div>

The majority of the epigenomic reports in hepatocellular carcinoma have focused on identifying novel differentially methylated drivers or passengers of the oncogenic process. Few reports have considered the technologies in place for clinical translation of newly identified biomarkers. The aim of this study was to identify epigenomic technologies that need only a small number of samples to discriminate HCC from non-HCC tissue, a basic requirement for biomarker development trials. To assess that potential, we used quantitative Methylation Specific PCR, oligonucleotide tiling arrays, and Methylation BeadChip assays. Concurrent global DNA hypomethylation, gene-specific hypermethylation, and chromatin alterations were observed as a hallmark of HCC. A global loss of promoter methylation was observed in HCC with the Illumina BeadChip assays and the Nimblegen oligonucleotide arrays. HCC samples had lower median methylation peak scores and a reduced number of significant promoter-wide methylated probes. Promoter hypermethylation of RASSF1A, SSBP2, and B4GALT1 quantified by qMSP had a sensitivity ranging from 38% to 52%, a specificity of 100%, and an AUC from 0.58 to 0.75. A panel combining these genes with HCC risk factors had a sensitivity of 87%, a specificity of 100%, and an AUC of 0.91.

While the molecular functions of miR-200 family have been deeply investigated, a role for these miRNAs as breast cancer biomarkers remains largely unexplored. In the attempt to clarify this, we profiled the miR-200 family members expression in a large cohort of breast cancer cases with a long follow-up (H-CSS cohort) and in TCGA-BRCA cohort. Overall, miR-200 family was found upregulated in breast tumors with respect to normal breast tissues while downregulated in more aggressive breast cancer molecular subtypes (i.e. Luminal B, HER2 and triple negative), consistently with their function as repressors of the epithelial-to-mesenchymal transition (EMT). In particular miR-141-3p was found differentially expressed in breast cancer molecular subtypes in both H-CSS and TCGA-BRCA cohorts, and the combined analysis of all miR-200 family members demonstrated a slight predictive accuracy on H-CSS cancer specific survival at 12 years (survival c-statistic: 0.646; 95%CI 0.538-0.754).

Abstract Background The hippocampus is involved in several complex diseases which display sex differences in their prevalence. Therefore, it can be hypothesized that sex‐specific genetic vulnerability in hippocampal formation might partially explain these differences. The aim of this study was to investigate whether the genetic predisposition to several complex diseases showed sex‐specific associations with the volumes of hippocampal subfields. Method A total of 1,071 cognitively unimpaired participants (45‐74 years) at increased risk for Alzheimer's disease (AD) (ALFA study), with available genotyping and neuroimaging data were included (62.6% women). Genetic predisposition was calculated through polygenic risk scores (PRSs) computed using the PRSice tool (v.1.25). A total of 33 PRSs were estimated from the most recent genome‐wide association meta‐analyses for several neurodegenerative, vascular and metabolic diseases, as well as for biomarkers of AD. Association analyses included the volumes of the hippocampal subfields quantified through high‐resolution 3D‐T1‐weighted magnetic resonance imaging scans and determined using Freesurfer (v.6.0). Sex‐stratified association analyses were estimated using linear regression models adjusted for age, education, and total intracranial volume. The threshold of significance was established by approximating the total number of effective tests. The resulting adjusted significance threshold was set at p <0.005. Result PRSs were equally distributed among women and men, sharing a common pattern of variability [Figure 1]. Both groups showed increased genetic susceptibility for abnormal levels of AD biomarkers and for vascular, brain, as well as sleep disorders. Association analyses showed significant sex differences associated to several hippocampal subfields [Figure 2]. Specifically, in women, increased genetic risk of multiple sclerosis and neurodegenerative diseases was associated with larger cornu ammonis 1 (CA1), hippocampal tail and molecular layer, as well as with increased volume of the whole hippocampus. In men, a raised genetic risk of cerebral infarction, epilepsy and both short and long duration of habitual sleep was associated with reduced volumes of CA1, molecular layer, subiculum, and dentate gyrus. Conclusion We provide evidence that genetic susceptibility differentially influences on hippocampal subfield volumes according to sex. This may explain sex‐specific patterns of neurodegeneration and biological vulnerabilities.

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