Publications

<p>'Supplementary Methods'</p>

Aberrant expression of some tumour suppressor genes and oncogenes by thymocytes had been involved in the development of primary thymic lymphomas induced by γ-irradiation, but genetic alterations affecting critical genes expressed by stromal cells have not been yet explored. This paper analyzes a series of such tumours induced in C57BL/6J and in F1 hybrids of BALB/c and C57BL/6J mouse strains. As expected, hystopathological analyses revealed profound disorganizations within the thymus with a poor demarcation of the cortical and medullar areas. Immunological and quantitative on-line RT–PCR analyses confirm that E-cadherin ( Cdh1 ) is essentially expressed by stromal cells of the thymus, while evidencing that the expression of this gene is significantly reduced in all tumours. In addition, and contrary to what one would expect, N-cadherin ( Cdh2 ) that is exclusively expressed by stromal cells is likewise down-regulated in most of the thymic lymphomas. Although hypermethylation of the promoter region appears to be involved in the inactivation of Cdh2 in all tumours, additional epigenetic mechanisms mediated by repressors such as Snai1 may also play a role in Cdh1 silencing. These results represent the first reported case for tumour-associated gene alterations occurring not in the tumour cells per se, but in the stromal cells of primary thymic lymphomas. Additionally, since the expression of both genes is significantly up-regulated after a single high dose of γ-radiation, but remained unchanged in treated thymic-lymphoma-free-mice, epigenetic down-regulation of E- and N-cadherin appears to occur concomitantly with the progression towards the most advanced stages of γ-radiation-induced thymic lymphomas.

Univariate and multivariate logistic regression of the first CRC patient cohort. (XLSX 9 kb)

The transcription factor aryl hydrocarbon receptor (AhR) has relevant functions in cell proliferation. Interestingly, the AhR can either promote or inhibit proliferation depending on the cell phenotype. Although recent data reveal potential pathways for AhR signaling in cell proliferation, the mechanisms that regulate its activity in tumor cells remain unknown. Here, we have analyzed promoter hypermethylation as a potential mechanism controlling AhR expression in human tumor cells. AhR promoter CpG methylation was sporadic in a panel of 19 tumor cell lines except for the chronic myeloid leukemia (CML) K562 and the acute lymphoblastic leukemia (ALL) REH. When compared with normal lymphocytes, REH had very low constitutive AhR expression that could be attributed to promoter hypermethylation since treatment with the DNA demethylating agent 5-aza-2′-deoxycitidine (AZA) significantly increased AhR mRNA and protein. These results in leukemia-derived cell lines were further confirmed in primary ALL, where 33% of the patients (7/21) had AhR promoter hypermethylation. Chromatin immunoprecipitation (ChIP) showed that methylation impaired binding of the transcription factor Sp1 to the AhR promoter, thus providing a mechanism for AhR downregulation in REH cells. Therefore, promoter hypermethylation represents a novel epigenetic mechanism downregulating AhR activity in hematological malignancies such as ALL.

DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.

<p>Physical and Functional interactions of proteins encoded by 9q32-q33.1 genes expressed in PDOXs of this study</p>

Abstract Immune checkpoint blockade (ICB) approaches have changed the therapeutic landscape for many tumor types. However, half of cutaneous squamous cell carcinoma (cSCC) patients remain unresponsive or develop resistance. Here, we show that, during cSCC progression in male mice, cancer cells acquire epithelial/mesenchymal plasticity and change their immune checkpoint (IC) ligand profile according to their features, dictating the IC pathways involved in immune evasion. Epithelial cancer cells, through the PD-1/PD-L1 pathway, and mesenchymal cancer cells, through the CTLA-4/CD80 and TIGIT/CD155 pathways, differentially block antitumor immune responses and determine the response to ICB therapies. Accordingly, the anti-PD-L1/TIGIT combination is the most effective strategy for blocking the growth of cSCCs that contain both epithelial and mesenchymal cancer cells. The expression of E-cadherin/Vimentin/CD80/CD155 proteins in cSCC, HNSCC and melanoma patient samples predicts response to anti-PD-1/PD-L1 therapy. Collectively, our findings indicate that the selection of ICB therapies should take into account the epithelial/mesenchymal features of cancer cells.

Epigenetic deregulation is a critical event in human malignancies. Besides mutations in TERT promoter, found in 20% of dysplastic nodules, little is known about the key molecular alterations driving early hepatocarcinogenesis. The aims of this study were: 1) to analyze DNA methylation changes during the transition from preneoplastic lesions to early hepatocellular carcinoma (HCC), and 2) to identify candidate epigenetic gatekeepers in the transition between dysplasia and early tumors. Methylome profiling was done with Illumina HumanMethylation450 (485,000 CpG, 96% of known CpG islands). We evaluated differentially methylated CpG sites between groups using an F-test. To detect novel epigenetic gatekeepers we defined hypermethylation as a B value higher than 0.5. We profiled 144 fresh-frozen tissues from resection or liver transplant specimens and integrated these data with our previously report on DNA methylation in HCC (Villanueva et al. Hepatology 2015) resulting in: 16 normal liver, 139 cirrhosis, 8 dysplastic nodules and 227 HCC samples. Correlation between methylation and RNA expression (n= 361) was quantified with the Pearson’s coefficient. Patients were mostly male (76%), with a median age of 66, and with underlying liver disease mainly due to hepatitis C (43%) and B (23%). All tumors were early stage according to BCLC classification, including 43 cases below 2 cm in diameter. A phylo-epigenetic tree derived from the euclidean distances between differentially DNA methylated sites (n=421,997) reveals a gradient of methylation changes that spans normal, cirrhotic, dysplastic nodules and HCC. Epigenetic analysis confirmed closer proximity of dysplasia to HCC than to cirrhotic tissue. We validated aberrant methylation of previously reported HCC epidrivers within the unpublished dataset (e.g. EFBN2, TBX15) (Villanueva et al. Hepatology 2015). Focusing on CpG sites located in promoter regions (i.e., TSS200, TSS1500, 5’UTR, and 1st exon), we selected candidates hypermethylated in less than 1% of normal and cirrhotic tissue, in all high grade dysplastic nodules, and in more than 50% of small HCC nodules (<2 cm). When we integrated DNA methylation and gene expression we found a significant (all P<0.001) inverse correlation in TSPYL5 (r=-0.31), KCNA3 (r=-0.33), LDBH (r=-0.46) and SPINT2 (r=-0.43). Whole-genome DNA methylation profiles accurately discriminate between different histological lesions along the human hepatocarcinogenesis spectrum. We report novel epigenetic gatekeepers in the transition between dysplastic nodules and early HCC. Citation Format: Gabriela Hernandez Meza, Johann von Felden, Amanda Craig, Sergi Sayols, Anna Portela, Manel Esteller, Myron Schwartz, Vincenzo Mazzaferro, Josep Llovet, Augusto Villanueva. Genome-wide DNA methylation profiling reveals novel candidate epigenetic gatekeepers in hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 474.

There was an error published in J. Cell Sci. 116, 499–511.For the GST-Snail band-shift assay (right-hand panel) shown in Fig. 2, the incorrect data was inadvertently shown to represent the results of the competition with the 1000-fold unlabeled E-pal wild-type (wt) probe experiment (first lane on the left). Additionally, the indication of the removal of lanes was omitted in the original figure. The correct Fig. 2 is presented below. There are no changes to the figure legend, which is accurate. This error does not affect the conclusions of the study. The authors apologise to the readers for any confusion that this error might have caused.

<p>Multi-drug resistance phenotype was ruled out by the expression of MDR1 (left) and MDR2 (right) genes by real time PCR. RQ values calculated by the DDCt method. Karpas1718 (K1718) parental lines in grey and resistant in red. Data derived from two independent experiments; error bars represent standard deviation of the mean.</p>

Abstract The transcription factor activator protein-2α (AP-2α) has recently been implicated as a tumor suppressor protein that can be lost during tumor progression and that exhibits growth-inhibitory properties when overexpressed in cancer cell lines. We now demonstrate that hypermethylation of a discrete 5′ region within a promoter CpG island of the gene is associated in breast cancer with the loss of AP-2α expression. Multiple CpG sites within the island become hypermethylated during breast cancer evolution. However, only hypermethylation of the most CpG-rich region, a small, ∼300-bp area at the 3′ end of exon 1, fully distinguishes neoplastic from normal breast tissue and correlates with transcriptional silencing. In cell culture, silenced AP-2α, associated with exon 1 hypermethylation, is re-expressed by 5-aza-2′deoxycytidine resulting in the restoration of a functional DNA sequence-specific binding protein. In vivo, as detected by a very sensitive nested PCR approach, methylation of the discrete AP-2α exon 1 region does not occur in normal breast epithelium and occurs in only 3 (16%) of 19 ductal carcinoma in situ (DCIS) lesions, but is present in 12 (75%) of 16 invasive breast tumors (P < 0.001; DCIS versus invasive cancers). Tumors unmethylated for this region expressed AP-2α protein throughout, whereas tumors with hypermethylation showed large areas of loss. Our studies then determine that hypermethylation of a small region of a CpG island correlates with silencing of AP-2α in breast cancer and suggest that inactivation of this gene could be a factor in, and a useful marker for, the progression of DCIS lesions.

Methyl CpG binding protein 2 (MeCP2) is a chromosomal protein of the brain, very abundant especially in neurons, where it plays an important role in the regulation of gene expression. Hence it has the potential to be affected by the mammalian circadian cycle. We performed expression analyses of mice brain frontal cortices obtained at different time points and we found that the levels of MeCP2 are altered circadianly, affecting overall organization of brain chromatin and resulting in a circadian-dependent regulation of well-stablished MeCP2 target genes. Furthermore, this data suggests that alterations of MeCP2 can be responsible for the sleeping disorders arising from pathological stages, such as in autism and Rett syndrome.

High-throughput genomic technologies are increasingly used in personalized cancer medicine. However, computational tools to maximize the use of scarce tissues combining distinct molecular layers are needed. Here we present a refined strategy, based on the R-package 'conumee', to better predict somatic copy number alterations (SCNA) from deoxyribonucleic acid (DNA) methylation arrays. Our approach, termed hereafter as 'conumee-KCN', improves SCNA prediction by incorporating tumor purity and dynamic thresholding. We trained our algorithm using paired DNA methylation and SNP Array 6.0 data from The Cancer Genome Atlas samples and confirmed its performance in cancer cell lines. Most importantly, the application of our approach in cancers of unknown primary identified amplified potentially actionable targets that were experimentally validated by Fluorescence in situ hybridization and immunostaining, reaching 100% specificity and 93.3% sensitivity.