Publications

<div>Abstract<p>Long-range oncogenic enhancers play an important role in cancer. Yet, whether similar regulation of tumor suppressor genes is relevant remains unclear. Loss of expression of PTEN is associated with the pathogenesis of various cancers, including T-cell acute lymphoblastic leukemia (T-ALL). Here, we identify a highly conserved distal enhancer (PE) that interacts with the <i>PTEN</i> promoter in multiple hematopoietic populations, including T cells, and acts as a hub of relevant transcription factors in T-ALL. Consistently, loss of PE leads to reduced <i>PTEN</i> levels in T-ALL cells. Moreover, PE-null mice show reduced <i>Pten</i> levels in thymocytes and accelerated development of NOTCH1-induced T-ALL. Furthermore, secondary loss of PE in established leukemias leads to accelerated progression and a gene expression signature driven by <i>Pten</i> loss. Finally, we uncovered recurrent deletions encompassing PE in T-ALL, which are associated with decreased <i>PTEN</i> levels. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in cancer.</p>Significance:<p>Here, we identify a <i>PTEN</i> enhancer that is recurrently deleted in patients with T-ALL. Loss of this enhancer leads to reduced PTEN levels in T cells together with accelerated generation and progression of NOTCH1-induced leukemia <i>in vivo</i>. These results uncover long-range regulation of tumor suppressor genes as a relevant mechanism in cancer.</p><p><i>This article is highlighted in the In This Issue feature, p. 1</i></p></div>

Transcriptional repression mechanisms have emerged as one of the crucial processes for the downregulation of E-cadherin expression during development and tumour progression. Recently, several E-cadherin transcriptional repressors have been characterized (Snail, E12/E47, ZEB-1 and SIP-1) and shown to act through an interaction with proximal E-boxes of the E-cadherin promoter. We have analyzed the participation of another member of the Snail family, Slug, and observed that it also behaves as a repressor of E-cadherin expression. Stable expression of Slug in MDCK cells leads to the full repression of E-cadherin at transcriptional level and triggers a complete epithelial to mesenchymal transition. Slug-induced repression of E-cadherin is mediated by its binding to proximal E-boxes, particularly to the E-pal element of the mouse promoter. Detailed analysis of the binding affinity of different repressors to the E-pal element indicates that Slug binds with lower affinity than Snail and E47 proteins. These results, together with the known expression patterns of these factors in embryonic development and carcinoma cell lines, support the idea that the in vivo action of the different factors in E-cadherinrepression can be modulated by their relative concentrations as well as by specific cellular or tumour contexts.

Colorectal cancer is the fourth cause of cancer-related deaths worldwide, though detection at early stages associates with good prognosis. Thus, there is a clear demand for novel non-invasive tests for the early detection of colorectal cancer and premalignant advanced adenomas, to be used in population-wide screening programs. Aberrant DNA methylation detected in liquid biopsies, such as serum circulating cell-free DNA (cfDNA), is a promising source of non-invasive biomarkers. This study aimed to assess the feasibility of using cfDNA pooled samples to identify potential serum methylation biomarkers for the detection of advanced colorectal neoplasia (colorectal cancer or advanced adenomas) using microarray-based technology. cfDNA was extracted from serum samples from 20 individuals with no colorectal findings, 20 patients with advanced adenomas, and 20 patients with colorectal cancer (stages I and II). Two pooled samples were prepared for each pathological group using equal amounts of cfDNA from 10 individuals, sex-, age-, and recruitment hospital-matched. We measured the methylation levels of 866,836 CpG positions across the genome using the MethylationEPIC array. Pooled serum cfDNA methylation data meets the quality requirements. The proportion of detected CpG in all pools (> 99% with detection p value < 0.01) exceeded Illumina Infinium methylation data quality metrics of the number of sites detected. The differential methylation analysis revealed 1384 CpG sites (5% false discovery rate) with at least 10% difference in the methylation level between no colorectal findings controls and advanced neoplasia, the majority of which were hypomethylated. Unsupervised clustering showed that cfDNA methylation patterns can distinguish advanced neoplasia from healthy controls, as well as separate tumor tissue from healthy mucosa in an independent dataset. We also observed that advanced adenomas and stage I/II colorectal cancer methylation profiles, grouped as advanced neoplasia, are largely homogenous and clustered close together. This preliminary study shows the viability of microarray-based methylation biomarker discovery using pooled serum cfDNA samples as an alternative approach to tissue specimens. Our strategy sets an open door for deciphering new non-invasive biomarkers not only for colorectal cancer detection, but also for other types of cancers.

Abstract Background Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome may contribute to matrix metalloprotease (MMP) dysregulation given their key role in invasion, which is the first step of the metastatic process. Methods We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with invasion. We stably disrupted the CCCTC-Binding Factor (CTCF) binding site of a single insulator element in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. Our findings were then also tested in a ductal carcinoma in situ (DCIS) cohort. Results We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was strongly associated with worse relapse-free (HR = 1.57 [1.06 − 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 − 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability. Finally, we observed that the imbalance in the MMP expression predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p &lt; 0.01). Conclusion Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.

&lt;p&gt;Impact of some RNAi clones on cisplatin toxicity in Caenorhabditis elegans adults&lt;/p&gt;

The molecular mechanisms responsible for the pathophysiological traits of type 2 diabetes are incompletely understood. Here we have performed transcriptomic analysis in skeletal muscle, and plasma metabolomics from subjects with classical and early-onset forms of type 2 diabetes (T2D). Focused studies were also performed in tissues from ob/ob and db/db mice. We document that T2D, both early and late onset, are characterized by reduced muscle expression of genes involved in branched-chain amino acids (BCAA) metabolism. Weighted Co-expression Networks Analysis provided support to idea that the BCAA genes are relevant in the pathophysiology of type 2 diabetes, and that mitochondrial BCAA management is impaired in skeletal muscle from T2D patients. In diabetic mice model we detected alterations in skeletal muscle proteins involved in BCAA metabolism but not in obese mice. Metabolomic analysis revealed increased levels of branched-chain keto acids (BCKA), and BCAA in plasma of T2D patients, which may result from the disruption of muscle BCAA management. Our data support the view that inhibition of genes involved in BCAA handling in skeletal muscle takes place as part of the pathophysiology of type 2 diabetes, and this occurs both in early-onset and in classical type 2 diabetes.

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Polyamines are important regulators of cell growth and death. The polyamine modulated factor-1 (PMF-1) is involved in polyamine homeostasis. After identifying an enriched CpG island encompassing the PMF1 promoter, we aimed at evaluating the clinical relevance of PMF1 methylation in bladder cancer.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; The epigenetic silencing of PMF1 by hypermethylation was tested in bladder cancer cells (&lt;i&gt;n&lt;/i&gt; = 11) after azacytidine treatment. PMF1 methylation status was evaluated in 507 bladder tumors and 118 urinary specimens of bladder cancer patients and controls. PMF1 protein expression was analyzed by immunohistochemistry on tissue arrays containing bladder tumors for which PMF1 methylation was assessed (&lt;i&gt;n&lt;/i&gt; = 218).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; PMF1 hypermethylation was associated with gene expression loss, being restored &lt;i&gt;in vitro&lt;/i&gt; by a demethylating agent. An initial set of 101 primary frozen bladder tumors served to identify PMF1 hypermethylation in 88.1% of the cases. An independent set of 406 paraffin-embedded tumors also revealed a high PMF1 methylation rate (77.6%). PMF1 methylation was significantly associated with increasing stage (&lt;i&gt;P&lt;/i&gt; = 0.025). Immunohistochemical analyses revealed that PMF1 methylation was associated with cytoplasmic PMF1 expression loss (&lt;i&gt;P&lt;/i&gt; = 0.032). PMF1 protein expression patterns were significantly associated with stage (&lt;i&gt;P&lt;/i&gt; &lt; 0.001), grade (&lt;i&gt;P&lt;/i&gt; &lt; 0.001), and poor overall survival using univariate (&lt;i&gt;P&lt;/i&gt; &lt; 0.001) and multivariate (&lt;i&gt;P&lt;/i&gt; = 0.011) analyses. Moreover, PMF1 methylation in urinary specimens distinguished bladder cancer patients from controls (area under the curve = 0.800).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusion:&lt;/b&gt; PMF1 was identified to be epigenetically modified in bladder cancer. The association of PMF1 methylation with tumor progression and its diagnostic ability using urinary specimens support including PMF1 assessment for the clinical management of bladder cancer patients.&lt;/p&gt;&lt;/div&gt;

Autism spectrum disorders are associated with defects in social response and communication that often occur in the context of intellectual disability. Rett syndrome is one example in which epilepsy, motor impairment, and motor disturbance may co-occur. Mutations in histone demethylases are known to occur in several of these syndromes. Herein, we aimed to identify whether mutations in the candidate histone demethylase JMJD1C (jumonji domain containing 1C) are implicated in these disorders.We performed the mutational and functional analysis of JMJD1C in 215 cases of autism spectrum disorders, intellectual disability, and Rett syndrome without a known genetic defect.We found seven JMJD1C variants that were not present in any control sample (~ 6,000) and caused an amino acid change involving a different functional group. From these, two de novo JMJD1C germline mutations were identified in a case of Rett syndrome and in a patient with intellectual disability. The functional study of the JMJD1C mutant Rett syndrome patient demonstrated that the altered protein had abnormal subcellular localization, diminished activity to demethylate the DNA damage-response protein MDC1, and reduced binding to MECP2. We confirmed that JMJD1C protein is widely expressed in brain regions and that its depletion compromises dendritic activity.Our findings indicate that mutations in JMJD1C contribute to the development of Rett syndrome and intellectual disability.Genet Med 18 1, 378-385.

BACKGROUND: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage (BAL) fluid. METHODS: Tumor-specific oncogene mutations, CpG-island methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. RESULTS: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. CONCLUSION: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC.

Supplementary Figures from NID2 and HOXA9 Promoter Hypermethylation as Biomarkers for Prevention and Early Detection in Oral Cavity Squamous Cell Carcinoma Tissues and Saliva

&lt;p&gt;Multi-omics analyses. Output tables of the moderated t-tests comparing transcriptome, microRNA and methylation profiles of resistant and parental. Output results of Gene Set Enrichment Analysis comparing GEP data of resistant and parental.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; O&lt;sup&gt;6&lt;/sup&gt;-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein removing mutagenic and cytotoxic adducts from O&lt;sup&gt;6&lt;/sup&gt;-guanine in DNA. Approximately 40% of colorectal cancers (CRC) display MGMT deficiency due to the promoter hypermethylation leading to silencing of the gene. Alkylating agents, such as dacarbazine, exert their antitumor activity by DNA methylation at the O&lt;sup&gt;6&lt;/sup&gt;-guanine site, inducing base pair mismatch; therefore, activity of dacarbazine could be enhanced in CRCs lacking &lt;i&gt;MGMT&lt;/i&gt;. We conducted a phase II study with dacarbazine in CRCs who had failed standard therapies (oxaliplatin, irinotecan, fluoropyrimidines, and cetuximab or panitumumab if &lt;i&gt;KRAS&lt;/i&gt; wild-type).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; All patients had tumor tissue assessed for &lt;i&gt;MGMT&lt;/i&gt; as promoter hypermethylation in double-blind for treatment outcome. Patients received dacarbazine 250 mg/m&lt;sup&gt;2&lt;/sup&gt; intravenously every day for four consecutive days, every 21 days, until progressive disease or intolerable toxicity. We used a Simon two-stage design to determine whether the overall response rate would be 10% or more. Secondary endpoints included association of response, progression-free survival, and disease control rate with &lt;i&gt;MGMT&lt;/i&gt; status.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Sixty-eight patients were enrolled from May 2011 to March 2012. Patients received a median of three cycles of dacarbazine (range 1–12). Grades 3 and 4 toxicities included: fatigue (41%), nausea/vomiting (29%), constipation (25%), platelet count decrease (19%), and anemia (18%). Overall, two patients (3%) achieved partial response and eight patients (12%) had stable disease. Disease control rate (partial response + stable disease) was significantly associated with &lt;i&gt;MGMT&lt;/i&gt; promoter hypermethylation in the corresponding tumors.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusion:&lt;/b&gt; Objective clinical responses to dacarbazine in patients with metastatic CRC are confined to those tumors harboring epigenetic inactivation of the DNA repair enzyme MGMT. &lt;i&gt;Clin Cancer Res; 19(8); 2265–72. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;PDF File - 916K, Representative pyrograms in a healthy colonic mucosa sample for the markers analyzed.&lt;/p&gt;