Publications

<div>Abstract<p><b>Purpose:</b> Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.</p><p><b>Experimental Design:</b> We used downregulation and methylation of <i>RBP1,</i> as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.</p><p><b>Results:</b> One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in <i>IDH1</i>. A variant in <i>GOT2</i>, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in <i>SDHC</i> in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline <i>IDH3B</i> mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.</p><p><b>Conclusions:</b> This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. <i>Clin Cancer Res; 23(20); 6315–24. ©2017 AACR</i>.</p></div>

Disruption of microRNA (miRNA) expression patterns is now being recognized as a hallmark of human cancer. The causes of these altered profiles are diverse, and, among them, we found the existence of defects in the miRNA processing machinery. However, little is known about how these alterations affect the biology of the underlying tumors. Herein, we show that colorectal cancer cells with an impairment in DICER1, a major miRNA biogenesis gene, undergo enrichment of tumor stemness features and an epithelial-to-mesenchymal transition. These phenotypes are associated with the downregulation of miRNAs, such as miR-34a, miR-126 and those of the miR-200 family, that target critical coding genes in these pathways. Most importantly, DICER1 impairment also induces the acquisition of a greater capacity for tumor initiation and metastasis, two properties associated with cancer stem cells.

The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses.

Adipose tissue dysfunction, particularly the visceral (VAT) compartment, has been proposed to play a relevant role in colorectal cancer (CRC) development and progression. Epigenetic mechanisms could be involved in this association. The current study aimed to evaluate if specific epigenetic marks in VAT are associated with colorectal cancer (CRC) to identify epigenetic hallmarks of adipose tissue-related CRC. Epigenome-wide DNA methylation was evaluated in VAT from 25 healthy participants and 29 CRC patients, using the Infinium HumanMethylation450K BeadChip. The epigenome-wide methylation analysis identified 170,184 sites able to perfectly separate the CRC and healthy samples. The differentially methylated CpG sites (DMCpGs) showed a global trend for increased methylated levels in CRC with respect to healthy group. Most of the genes encoded by the DMCpGs belonged to metabolic pathways and cell cycle, insulin resistance, and adipocytokine signalling, as well as tumoural transformation processes. In gene-specific analyses, involved genes biologically relevant for the development of CRC include PTPRN2, MAD1L1, TNXB, DIP2C, INPP5A, HDCA4, PRDM16, RPTOR, ATP11A, TBCD, PABPC3, and IER2. The methylation level of some of them showed a discriminatory capacity for detecting CRC higher than 90%, showing IER2 to have the highest capacity. This study reveals that a specific methylation pattern of VAT is associated with CRC. Some of the epigenetic marks identified could provide useful tools for the prediction and personalized treatment of CRC connected to excess adiposity.

Abstract Introduction Endothelin (EDN) signalling plays a crucial role in cell differentiation, proliferation and migration processes. There is compelling evidence that altered EDN signalling is involved in carcinogenesis by modulating cell survival and promoting invasiveness. To date, most reports have focused on the oncogenic potential of EDN1 and EDN2, both of which are overexpressed in various tumour entities. Here, we aimed at a first comprehensive analysis on EDN3 expression and its implication in human breast cancer. Methods EDN3 mRNA expression was assessed by Northern blotting in normal human tissues (n = 9) as well as in matched pairs of normal and tumourous tissues from breast specimens (n = 50). EDN3 mRNA expression in breast cancer was further validated by real-time polymerase chain reaction (PCR) (n = 77). A tissue microarray was used to study EDN3 protein expression in breast carcinoma (n = 150) and normal breast epithelium (n = 44). EDN3 promoter methylation was analysed by methylation-specific PCR in breast cell lines (n = 6) before and after demethylating treatment, normal breast tissues (n = 17) and primary breast carcinomas (n = 128). EDN3 expression and methylation data were statistically correlated with clinical patient characteristics and patient outcome. Results Loss of EDN3 mRNA expression in breast cancer, as initially detected by array-based expression profiling, could be confirmed by Northern blot analysis (> 2-fold loss in 96%) and real-time PCR (> 2-fold loss in 78%). Attenuated EDN3 expression in breast carcinoma was also evident at the protein level (45%) in association with adverse patient outcome in univariate ( P = 0.022) and multivariate (hazard ratio 2.0; P = 0.025) analyses. Hypermethylation of the EDN3 promoter could be identified as the predominant mechanism leading to gene silencing. Reversion of the epigenetic lock by 5-aza-2'-deoxycytidine and trichostatin A resulted in EDN3 mRNA re-expression in vitro . Furthermore, EDN3 promoter hypermethylation was detected in 70% of primary breast carcinomas with significant association to loss of EDN3 mRNA expression ( P = 0.005), whilst normal matched breast tissues revealed no EDN3 promoter methylation. Conclusions EDN3 is a frequent target of epigenetic inactivation in human breast cancer, potentially contributing to imbalanced EDN signalling commonly found in this disease. The clinical implication supports the view that EDN3, in contrast to EDN1 and EDN2, may act as natural tumour suppressor in the human mammary gland.

<p>Panel of kinases analyzed in the Phospho Flow experiments of parental and resistant cells by flow cytometry.</p>

3728 Background: DNA methylation profiles in cancer cells are characterized by an overall decrease in the level of 5-methylcytosine and regional hypermethylation on particular CpG islands. We have addressed the concurrent study of DNA hyper- and hypomethylation and the analysis of its possible correlates with other features in a series of human colorectal cancers. Methods: DNA methylation profiles in sporadic colorectal carcinomas (n=98), synchronous adenoma-carcinoma pairs (n=11) and their paired normal mucosa were analyzed by using the Amplification of Inter-Methylated Sites (AIMS) method. A total of 208 anonymous methylated tags were evaluated for differential methylation. Global indices of hyper- and hypomethylation were calculated. Also MSI, K-ras, p53 mutations and hypermethylation at specific genes were assessed. Results: All adenomas and carcinomas displayed altered patterns of DNA methylation in reference to the normal tissue. In average, 24% of the represented tags were differentially methylated in carcinomas in regard to the normal pair with an overall prevalence of hypomethylations (14.6%) to hypermethylations (9.6%). Carcinomas exhibited higher (9.6%) levels of hypermethylation than adenomas (7.1%) but similar levels of hypomethylation. Hypermethylation correlated with female sex, advanced tumor stage and number of hypermethylated genes. Hierarchical cluster analysis revealed two main patterns of DNA methylation that were associated to particular mutational spectra in the K-ras and the p53 genes. In both groups correlates of hypomethylation or hypermethylation with survival were observed. Discussion: DNA hypermethylation and hypomethylation appear to play different roles in colorectal tumor progression and correlate with overall survival in subgroups with specific genetic and epigenetic signatures. No significant financial relationships to disclose.

APOE-ε4 is the major genetic risk factor for sporadic Alzheimer's disease (AD). In patients, its presence relates with cognitive symptoms severity and increased hippocampal degeneration. Since AD develops slowly, with a preclinical phase that can last for decades, a critical question is whether healthy individuals with higher genetic risk for AD present early neuroimaging signatures of the disease. However, previous studies investigating the impact of APOE-ε4 on grey matter (GM) volumes in cognitively normal individuals have so far shown inconsistent findings, mainly because of poor representation of the ε4-homozygotes in the surveyed samples. We analyzed regional grey matter (GM) volumes using optimized voxel-based morphometry in a sample of 526 middle-aged healthy individuals hosting an unprecedented number of ε4-homozygotes for a single-site study (254 non-carriers, 207 heterozygotes and 65 homozygotes). Compared to non-carriers, ε4-carriers showed reduced GM volumes in several brain regions including the bilateral hippocampus, caudate, and precuneus. We also detected regions displaying increased volume in ε4-homozygotes compared to both non-carriers and ε4-heterozygotes. Finally, we found a significant interaction between APOE genotype and age in target regions which play a critical role in AD pathophysiology, indicating that APOEstatus impacts on the lifespan trajectories of cortical GM volumes. Our data suggest that APOE-ε4 may exert its liability effect by altering brain morphology already in healthy individuals. These results enhance our understanding of how cortical degeneration may initiate in AD and therefore are insightful for developing targeted strategies, which shall delay the onset or slow-down the progression of the disease. Main effects of APOE genotype on grey matter (GM) volumes in 526 middle-aged healthy adults. A) Mean-centered bar plots showing GM volumetric differences among the five APOE genotype groups. Spatial coordinates are given in the Montral Neurological Institute (MNI) standard space. B) Statistical parametric maps displaying main effects of APOE genotype on GM volumes. The bilateral hippocampus and thalamus are represented in cold and warm colors, respectively. Reduced regional grey matter (GM) volumes in APOE-ε4 carriers compared to non-carriers. A) Mean-centered bar plots of contrast estimates. Compared to non-carriers, ε4-carriers show significantly reduced regional GM volumes in a gene dose-dependent manner. B) Mean-centered bar plots of contrast estimates. Significantly reduced GM volumes in ε4-carriers compared to non-carriers in regions showing no dose-dependent effects. C) Statistical parametric maps of brain regions where ε4-carriers displayed significant volumetric reductions compared to non-carriers (P<0.001). Increased regional grey matter (GM) volumes in APOE-ε4 homozygotes. A) Mean-centered bar plots showing significantly increased regional GM volumes in ε4-homozygotes compared to both heterozygotes and non-carriers. B) Statistical parametric maps of brain regions where ε4-homozygotes display significant volumetric increases reported in (A). Interaction between APOE status and age in determining lifespan trajectories of regional grey matter (GM) volumes. Compared to both non-carriers and heterozygotes, the ε4-homozygotes showed significantly reduced age-related GM volumes in the right hippocampus (A) and right lingual gyrus (B). By contrast, in the left thalamus (C), left temporal pole (D) and right superior frontal cortex (E), the ε4-homozygotes displayed increased age-related GM volumes compared to both non-carriers and heterozygotes.

Abstract Purpose: Diffuse Intrinsic Pontine Glioma (DIPG), is a very aggressive pediatric cancer with poor overall survival and no effective treatment. Despite numerous clinical trials, the overall survival remains at 9 months after diagnosis and only radiotherapy has shown a relative efficacy. Therefore, it is primordial to increase our understanding of the biology of DIPG tumors, as well as finding new healthcare strategies to tackle this pediatric disease. Recently, we discovered that NQO1, a stress-related protein, was overexpressed in 3 out of 6 DIPG patient-derived cell lines, as well as in half of the primary tissues. Interestingly, NQO1 overexpression can be targeted by substrates that induce an excessive oxidative stress into the cells that finally push them to apoptosis, while normal surrounding tissues with basal expression of NQO1 would be keep safe. Experimental procedure: This discovery prompted us to test in in vitro and in vivo assays a promising drug named isobutyl-deoxynyboquinone (IB-DNQ): a NQO1 bioactivatable substrate. We first tested the drug response of the 6 cell lines before testing it on orthotopic xenograft mouse models. We also investigated by which mechanism NQO1 is regulated in DIPG. Finally, we performed experiments in order to decipher by which mechanism NQO1 is regulated in DIPG. Results: We saw that NQO1 overexpressing cells are very sensitive to the drug, compared to the cell lines with normal expression. Moreover, we started to validate the use of IB-DNQ in vivo using a DIPG cell line treated or not (mock) with IB-DNQ and also a NQO1-knockdown model in orthotopic xenograft mice models, confirming that IB-DNQ crosses the blood brain barrier and increases the overall survival. NQO1 increased expression in DIPG is surprisingly not due to a NRF2-mediated transcriptional regulation as we did not observe a correlation between NQO1 transcript abundance and protein expression in DIPG cell lines, indicating that NRF2 pathway could not be implicated, but translational or post-translational regulation could be operating. We performed a polysome profiling of 4 patient-derived cell lines, a protein stability assay and a large scale proteome for the 6 cell lines (pending results). Conclusion: Our preliminary results, including drug efficacy, are very encouraging for the development of this new therapeutic in DIPG. This discovery represents a promising opportunity to tackle this devastating disease and a new hope for the patients and their families. Citation Format: Maxime Henri Janin, Vanessa Ortiz Barahona, Pere Llinas Arias, Carolina De La Torre, Angel Montero Carcaboso, Andres Morales La Madrid, Paul Hergenrother, Manel Esteller. Positioning the NQO1-bioactivatable drug isobutyl-deoxynyboquinone in diffuse intrinsic pontine glioma (DIPG): an exceptional therapeutic opportunity in pediatric brain tumor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5416.

Abstract Background: Cyclooxygenase 2 (COX-2) overexpression is a frequent but not universal event in colorectal cancer. It has been suggested that COX-2 protein expression is reduced in colorectal cancer with a defective mismatch repair (MMR) system, a phenomenon commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC) but also present in up to 15% of sporadic tumors. Aim: To assess COX-2 expression in a large series of fully characterized colorectal cancer patients with respect to the MMR system and to dissect the mechanisms responsible for altered COX-2 expression in this setting. Patients and Methods: MMR-deficient colorectal cancer were identified in a nationwide, prospective, multicenter study (EPICOLON project). Control MMR-proficient colorectal cancer patients were randomly selected. COX-2 expression was evaluated by immunohistochemistry. Personal and familial characteristics, as well as MSH2/MLH1 expression and germ line mutations, were evaluated. Results: One hundred fifty-three patients, 46 with MMR deficiency and 107 with MMR proficiency, were included in the analysis. Overall, tumor COX-2 overexpression was observed in 107 patients (70%). COX-2 overexpression was observed in 85 patients (79%) with a MMR-proficient system, but only in 22 patients (48%) with a MMR-deficient colorectal cancer (P &amp;lt; 0.001). The lack of COX-2 overexpression was independently associated with a MMR-deficient system (odds ratio, 3.89; 95% confidence interval, 1.78-8.51; P = 0.001) and a poor degree of differentiation (OR, 3.83; 95% CI, 1.30-11.31; P = 0.015). In the subset of patients with a MMR-deficient colorectal cancer, lack of COX-2 overexpression correlated with a poor degree of differentiation, no fulfillment of Amsterdam II criteria, absence of MSH2/MLH1 germ line mutations, presence of tumor MSH2 expression, and lack of tumor MLH1 expression. CpG island promoter hypermethylation of COX2 was observed in 6 of 18 (33%) tumors lacking COX-2 expression in comparison with 2 of 28 (7%) tumors expressing this protein (P = 0.04). Conclusions: Up to half of MMR-deficient colorectal cancer do not show COX-2 overexpression, a fact observed almost exclusively in patients with sporadic forms. COX2 hypermethylation seems to be responsible for gene silencing in one third of them. These results suggest the potential utility of nonsteroidal anti-inflammatory drugs in HNPCC chemoprevention and may explain the lack of response of this approach in some sporadic tumors.

&lt;div&gt;Abstract&lt;p&gt;Differentially methylated oral squamous cell carcinoma (OSCC) biomarkers, identified &lt;i&gt;in vitro&lt;/i&gt; and validated in well-characterized surgical specimens, have shown poor clinical correlation in cohorts with different risk profiles.&lt;/p&gt;&lt;p&gt;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.&lt;/p&gt;&lt;p&gt;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.&lt;/p&gt;&lt;p&gt;Promoter methylation of &lt;i&gt;KIF1A&lt;/i&gt; (κ = 0.64), &lt;i&gt;HOXA9&lt;/i&gt; (κ = 0.60), &lt;i&gt;NID2&lt;/i&gt; (κ = 0.60), and &lt;i&gt;EDNRB&lt;/i&gt; (κ = 0.60) had a moderate to substantial agreement with clinical diagnosis in the discovery screen. &lt;i&gt;HOXA9&lt;/i&gt; had 68% sensitivity, 100% specificity, and a 0.81 Area Under the Curve (AUC). &lt;i&gt;NID2&lt;/i&gt; had 71% sensitivity, 100% specificity, and a 0.79 AUC. In the prevalence screen, &lt;i&gt;HOXA9&lt;/i&gt; (κ = 0.82) and &lt;i&gt;NID2&lt;/i&gt; (κ = 0.80) had an almost perfect agreement with histologic diagnosis. &lt;i&gt;HOXA9&lt;/i&gt; had 85% sensitivity, 97% specificity, and a 0.95 AUC. &lt;i&gt;NID2&lt;/i&gt; had 87% sensitivity, 95% specificity, and a 0.91 AUC. A &lt;i&gt;HOXA9&lt;/i&gt; and &lt;i&gt;NID2&lt;/i&gt; gene panel had 94% sensitivity, 97% specificity, and a 0.97 AUC. In saliva, from OSCC cases and controls, &lt;i&gt;HOXA9&lt;/i&gt; had 75% sensitivity, 53% specificity, and a 0.75 AUC. &lt;i&gt;NID2&lt;/i&gt; had 87% sensitivity, 21% specificity, and a 0.73 AUC.&lt;/p&gt;&lt;p&gt;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. &lt;i&gt;Cancer Prev Res; 4(7); 1061–72. ©2011 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Background & Aims Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary hepatic cancer that develops in children and young adults without cirrhosis. Little is known about its pathogenesis, and it can be treated only with surgery. We performed an integrative genomic analysis of a large series of patients with FLC to identify associated genetic factors. Methods By using 78 clinically annotated FLC samples, we performed whole-transcriptome (n = 58), single-nucleotide polymorphism array (n = 41), and next-generation sequencing (n = 48) analyses; we also assessed the prevalence of the DNAJB1−PRKACA fusion transcript associated with this cancer (n = 73). We performed class discovery using non-negative matrix factorization, and functional annotation using gene-set enrichment analyses, nearest template prediction, ingenuity pathway analyses, and immunohistochemistry. The genomic identification of significant targets in a cancer algorithm was used to identify chromosomal aberrations, MuTect and VarScan2 were used to identify somatic mutations, and the random survival forest was used to determine patient prognoses. Findings were validated in an independent cohort. Results Unsupervised gene expression clustering showed 3 robust molecular classes of tumors: the proliferation class (51% of samples) had altered expression of genes that regulate proliferation and mammalian target of rapamycin signaling activation; the inflammation class (26% of samples) had altered expression of genes that regulate inflammation and cytokine enriched production; and the unannotated class (23% of samples) had a gene expression signature that was not associated previously with liver tumors. Expression of genes that regulate neuroendocrine function, as well as histologic markers of cholangiocytes and hepatocytes, were detected in all 3 classes. FLCs had few copy number variations; the most frequent were focal amplification at 8q24.3 (in 12.5% of samples), and deletions at 19p13 (in 28% of samples) and 22q13.32 (in 25% of samples). The DNAJB1−PRKACA fusion transcript was detected in 79% of samples. FLC samples also contained mutations in cancer-related genes such as BRCA2 (in 4.2% of samples), which are uncommon in liver neoplasms. However, FLCs did not contain mutations most commonly detected in liver cancers. We identified an 8-gene signature that predicted survival of patients with FLC. Conclusions In a genomic analysis of 78 FLC samples, we identified 3 classes based on gene expression profiles. FLCs contain mutations and chromosomal aberrations not previously associated with liver cancer, and almost 80% contain the DNAJB1−PRKACA fusion transcript. By using this information, we identified a gene signature that is associated with patient survival time.

&lt;p&gt;Supplementary Table 2. Genes that significantly change expression upon KAT6B depletion. Normalized intensities from the array in each sample and log Fold Changes from the analysis are depicted.&lt;/p&gt;

Supplementary Tables from A Mouse Skin Multistage Carcinogenesis Model Reflects the Aberrant DNA Methylation Patterns of Human Tumors

Epigenetic deregulation has emerged as a driver in human malignancies. There is no clear understanding of the epigenetic alterations in hepatocellular carcinoma (HCC) and of the potential role of DNA methylation markers as prognostic biomarkers. Analysis of tumor tissue from 304 patients with HCC treated with surgical resection allowed us to generate a methylation-based prognostic signature using a training-validation scheme. Methylome profiling was done with the Illumina HumanMethylation450 array (Illumina, Inc., San Diego, CA), which covers 96% of known cytosine-phosphate-guanine (CpG) islands and 485,000 CpG, and transcriptome profiling was performed with Affymetrix Human Genome U219 Plate (Affymetrix, Inc., Santa Clara, CA) and miRNA Chip 2.0. Random survival forests enabled us to generate a methylation signature based on 36 methylation probes. We computed a risk score of mortality for each individual that accurately discriminated patient survival both in the training (221 patients; 47% hepatitis C-related HCC) and validation sets (n = 83; 47% alcohol-related HCC). This signature correlated with known predictors of poor outcome and retained independent prognostic capacity of survival along with multinodularity and platelet count. The subset of patients identified by this signature was enriched in the molecular subclass of proliferation with progenitor cell features. The study confirmed a high prevalence of genes known to be deregulated by aberrant methylation in HCC (e.g., Ras association [RalGDS/AF-6] domain family member 1, insulin-like growth factor 2, and adenomatous polyposis coli) and other solid tumors (e.g., NOTCH3) and describes potential candidate epidrivers (e.g., septin 9 and ephrin B2).A validated signature of 36 DNA methylation markers accurately predicts poor survival in patients with HCC. Patients with this methylation profile harbor messenger RNA-based signatures indicating tumors with progenitor cell features.