Publications

<p>Expression levels of surface ERBB4 and pERBB4 was measured by FACS (A) and immunoblotting (B) in Karpas1718 parental and resistant. (A) Two independent experiments were performed: exp1 (left panel) and exp2 (right panel). Density plots show the median MFI values of two replicates from each experiment: negative control (dotted black), parental (grey), resistant (red). Results are presented from Welch Two Sample test performed on MFI values of the two experiments. (B) Immunobloting for total (grey) and phospho (red) ERBB4 in parental (left) and resistant (right) Karpas1718 cells. Representative image of two independent experiments. Data on the barplot represents the mean of two experiments, errors bars represent standard deviation of the mean. * for p<0.05 from a t-test.</p>

Abstract Prostate cancer (PCa) is the most common noncutaneous malignancy and a leading cause of cancer death among men. Clinically localized disease might be successfully treated whereas disseminated disease remains mostly lethal. During prostate cancer development and progression, tumor cells undergo abnormal epigenetic modifications, including DNA methylation alterations. DNA methylation is the most studied epigenetic alteration in human cancer, and is associated with transcriptional repression of both coding and non-coding regions of the genome. Aberrant DNA methylation is gaining force in the fields of cancer risk assessment, diagnosis and therapy monitoring in different cancer types, including prostate cancer. Importantly, aberrant hypermethylation is widespread during neoplastic alteration of prostate cells, suggesting that restoration of a normal prostate epigenome through treatment with demethylating drugs could be clinically beneficial. The main aim of this work was to identify new epigenetically downregulated CpG's in PCa, using the 450K cytosine microarray-based approach, followed by validation in a larger set of clinical samples. We used the Infinium HumanMethylation450 BeadChip (Illumina) to consistently and significantly detect CpG methylation changes in prostate cancer tissues in comparison with morphological normal prostate tissue. The selected genomic regions were validated by pyrosequencing. The methylation data were subsequently confirmed at mRNA level using qRT-PCR. To prove the functional role of aberrant DNA methylation, we also performed a treatment with 5-Aza-2′-Deoxycytidine in PCa cell lines. Our work might provide new translational applications to the clinical management of PCa patients, based on of DNA methylation changes in PCa. Citation Format: Joao Ramalho-Carvalho, Rui Henrique, Sergi Sayols, Antonio Gomez, Carmen Jerónimo, Manel Esteller. Redefining the DNA methylome of prostate cancer using DNA methylation arrays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4252. doi:10.1158/1538-7445.AM2013-4252

<p>Mutational burden at diagnosis across patients and correlation between single-cell and bulk VAFs</p>

<p>Sequences of sgRNAs used for induction of ERBB4 transcription by CRISPR activation technique.</p>

Monozygotic (MZ) twins are partially concordant for most complex diseases, including autoimmune disorders. Whereas phenotypic concordance can be used to study heritability, discordance suggests the role of non-genetic factors. In autoimmune diseases, environmentally driven epigenetic changes are thought to contribute to their etiology. Here we report the first high-throughput and candidate sequence analyses of DNA methylation to investigate discordance for autoimmune disease in twins. We used a cohort of MZ twins discordant for three diseases whose clinical signs often overlap: systemic lupus erythematosus (SLE), rheumatoid arthritis, and dermatomyositis. Only MZ twins discordant for SLE featured widespread changes in the DNA methylation status of a significant number of genes. Gene ontology analysis revealed enrichment in categories associated with immune function. Individual analysis confirmed the existence of DNA methylation and expression changes in genes relevant to SLE pathogenesis. These changes occurred in parallel with a global decrease in the 5-methylcytosine content that was concomitantly accompanied with changes in DNA methylation and expression levels of ribosomal RNA genes, although no changes in repetitive sequences were found. Our findings not only identify potentially relevant DNA methylation markers for the clinical characterization of SLE patients but also support the notion that epigenetic changes may be critical in the clinical manifestations of autoimmune disease.

Gene silencing by DNA hypermethylation of CpG islands is a well-characterized phenomenon in cancer. The effect of hypomethylation in particular of non-CpG island genes is much less well described. By genome-wide screening, we identified 105 genes in microsatellite stable (MSS) colorectal adenocarcinomas with an inverse correlation (Spearman's ρ ≤ -0.40) between methylation and expression. Of these, 35 (33%) were hypomethylated non-CpG island genes and two of them, APOLD1 (Spearman's ρ = -0.82) and SRPX2 (Spearman's ρ = -0.80) were selected for further analyses. Hypomethylation of both genes were localized events not shared by adjacent genes. A set of 662 FFPE DNA samples not only confirmed that APOLD1 and SRPX2 are hypomethylated in CRC but also revealed hypomethylation to be significantly (p <0.01) associated with tumors being localized in the left side, CpG island methylator phenotype negative, MSS, BRAF wt, undifferentiated and of adenocarcinoma histosubtype. Demethylation experiments supported SRPX2 being epigenetically regulated via DNA methylation, whereas other mechanisms in addition to DNA methylation seem to be involved in the regulation of APOLD1. We further identified miR-149 as a potential novel post-transcriptional regulator of SRPX2. In carcinoma tissue, miR-149 was downregulated and inversely correlated to SRPX2 (ρ = -0.77). Furthermore, ectopic expression of miR-149 significantly reduced SRPX2 transcript levels. Our study highlights that in colorectal tumors, hypomethylation of non-CpG island-associated promoters deregulate gene expression nearly as frequent as do CpG-island hypermethylation. The hypomethylation of SRPX2 is focal and not part of a large block. Furthermore, it often translates to an increased expression level, which may be modulated by miR-149.

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3′UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT–qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

Background & aims Agents that induce an immune response against tumors by altering T-cell regulation have increased survival times of patients with advanced-stage tumors, such as melanoma or lung cancer. We aimed to characterize molecular features of immune cells that infiltrate hepatocellular carcinomas (HCCs) to determine whether these types of agents might be effective against liver tumors. Methods We analyzed HCC samples from 956 patients. We separated gene expression profiles from tumor, stromal, and immune cells using a non-negative matrix factorization algorithm. We then analyzed the gene expression pattern of inflammatory cells in HCC tumor samples. We correlated expression patterns with the presence of immune cell infiltrates and immune regulatory molecules, determined by pathology and immunohistochemical analyses, in a training set of 228 HCC samples. We validated the correlation in a validation set of 728 tumor samples. Using data from 190 tumors in the Cancer Genome Atlas, we correlated immune cell gene expression profiles with numbers of chromosomal aberrations (based on single-nucleotide polymorphism array) and mutations (exome sequence data). Results We found approximately 25% of HCCs to have markers of an inflammatory response, with high expression levels of the CD274 molecule (programmed death-ligand 1) and programmed cell death 1, markers of cytolytic activity, and fewer chromosomal aberrations. We called this group of tumors the Immune class. It contained 2 subtypes, characterized by markers of an adaptive T-cell response or exhausted immune response. The exhausted immune response subclass expressed many genes regulated by transforming growth factor beta 1 that mediate immunosuppression. We did not observe any differences in numbers of mutations or expression of tumor antigens between the immune-specific class and other HCCs. Conclusions In an analysis of HCC samples from 956 patients, we found almost 25% to express markers of an inflammatory response. We identified 2 subclasses, characterized by adaptive or exhausted immune responses. These findings indicate that some HCCs might be susceptible to therapeutic agents designed to block the regulatory pathways in T cells, such as programmed death-ligand 1, programmed cell death 1, or transforming growth factor beta 1 inhibitors.

Physical exercise has been shown to induce epigenetic modifications with various health implications, directly affect DNA methylation (DNAm), as well as reverse the epigenetic age. Hence, we aimed to identify differential methylation changes and assess the epigenetic age in the saliva of 7-9-year-old school children following a 3-month integrated neuromuscular training (INT), as well as to explore if any of the methylation changes are in core genes. Core genes are defined as genes of high relevance and essential importance within the human genome. Forty children (17 boys and 23 girls) were recruited from schools in Girona, Spain, and allocated into control (N = 20) or INT (N = 20) group. The INT group performed a 3-month INT as a warm-up during the physical education (PE) classes, encompassing strength, coordination, dynamic stabilization, plyometrics, speed, and agility exercises, whereas the control group performed traditional warm-up activities, encompassing aerobic exercises that will prepare the cardiovascular system and increase the joint mobility for the upcoming effort during the class. Genome-wide DNAm analysis was performed with the Illumina 900 K microarray. Core genes were recognized based on the accomplishment of a rigorous and widely accepted 3-point criteria: participation in the enriched pathways, high connectivity (≥ 10), and target genes of key transcription factors. There were 1200 differentially methylated positions (DMPs) in the control group and 414 DMPs in the INT group (FDR < 0.05, p < 0.05, Aβ < |0.1|), suggesting a non-significant trend of epigenetic age acceleration in the control group (1.18 months, p > 0.05) and a non-significant 1-month decrease of the epigenetic age in the INT group (p > 0.05). The genes with DMPs in the control group showed low similarity between enriched pathways and low interconnectivity, encompassing distinct pathways, mostly development and growth-related. Additionally, no core genes were identified in the control group. Interestingly, the genes with DMPs in the INT group showed high similarity between enriched pathways and high interconnectivity, encompassing related pathways involving signaling mechanisms, as well as hormone and protein metabolism pathways. Moreover, 17 DMPs in the children from the INT group were in core genes. The main findings of the present study are suggesting an integrated response to the training stimulus in 7-9-year-old school children that performed a 3-month INT, including epigenetic modifications in 17 genes considered as core genes. Trial Registration: The study protocol was registered in the ISRCTN registry (ISRCTN16744821).