Publications

Low-level microsatellite instability in most colorectal carcinomas.

Twelve to 16% of colorectal cancers (CRCs) display a high degree of microsatellite instability (MSI-H), whereas most are believed to be microsatellite stable (MSS). The existence of a low degree of instability (MSI-L) group has also been proposed. By using the Bethesda panel of microsatellite markers, the microsatellite instability (MSI) status of CRCs can be determined. This set is recommended to distinguish between MSI-H and MSI-L/MSS. No definition for MSI-L has emerged. Most reports on MSI-L rely on the Bethesda panel, using 5-15markers. Tumors with more than 30% MSI are designated as MSI-H, but the lower limit for MSI-L is ambiguous. We hypothesized that if many markers are studied, almost all CRCs would show some MSI. It would be necessary to establish a cutoff level for MSI-L by showing that, above this cutoff level, tumors display molecular and/or clinical features different from those under the cutoff level. To perform this task, we analyzed 90 BAT26 stable CRC samples with 377 markers. MSI at 1-11 loci was observed in 71 (79%) of the 90 cases. K-RAS mutation, loss of heterozygosity, and MLH1 and MGMT hypermethylation analyses were performed, as well as clinical features being scrutinized, to examine possible differences between MSI-L and MSS tumors using all of the possible cutoff levels for MSI-L. Convincing differences between putative MSI-L and MSS groups were not observed. Our results show that the sensitivity of a typically used marker number to detect MSI-L is very low, and they suggest that MSS and MSI-L tumors have a common molecular background.

Erratum: The Polycomb group protein EZH2 directly controls DNA methylation

Data from Long-Range Epigenetic Silencing Associates with Deregulation of Ikaros Targets in Colorectal Cancer Cells

<div>Abstract<p>Transcription factors are common targets of epigenetic inactivation in human cancer. Promoter hypermethylation and subsequent silencing of transcription factors can lead to further deregulation of their targets. In this study, we explored the potential epigenetic deregulation in cancer of <i>Ikaros</i> family genes, which code for essential transcription factors in cell differentiation and exhibit genetic defects in hematologic neoplasias. Unexpectedly, our analysis revealed that <i>Ikaros</i> undergoes very specific promoter hypermethylation in colorectal cancer, including in all the cell lines studied and around 64% of primary colorectal adenocarcinomas, with increasing proportions in advanced Duke's stages. <i>Ikaros</i> hypermethylation occurred in the context of a novel long-range epigenetic silencing (LRES) region. Reintroduction of Ikaros in colorectal cancer cells, ChIP-chip analysis, and validation in primary samples led us to identify a number of direct targets that are possibly related with colorectal cancer progression. Our results not only provide the first evidence that LRES can have functional specific effects in cancer but also identify several deregulated Ikaros targets that may contribute to progression in colorectal adenocarcinoma. <i>Mol Cancer Res; 9(8); 1139–51. ©2011 AACR</i>.</p></div>

MiR-204 silencing in intraepithelial to invasive cutaneous squamous cell carcinoma progression

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and frequently progresses from an actinic keratosis (AK), a sun-induced keratinocyte intraepithelial neoplasia (KIN). Epigenetic mechanisms involved in the phenomenon of progression from AK to cSCC remain to be elicited. Expression of microRNAs in sun-exposed skin, AK and cSCC was analysed by Agilent microarrays. DNA methylation of miR-204 promoter was determined by bisulphite treatment and pyrosequencing. Identification of miR-204 targets and pathways was accomplished in HaCat cells. Immunofluorescence and immunohistochemistry were used to analyze STAT3 activation and PTPN11 expression in human biopsies. cSCCs display a marked downregulation of miR-204 expression when compared to AK. DNA methylation of miR-204 promoter was identified as one of the repressive mechanisms that accounts for miR-204 silencing in cSCC. In HaCaT cells miR-204 inhibits STAT3 and favours the MAPK signaling pathway, likely acting through PTPN11, a nuclear tyrosine phosphatase that is a direct miR-204 target. In non-peritumoral AK lesions, activated STAT3, as detected by pY705-STAT3 immunofluorescence, is retained in the membrane and cytoplasm compartments, whereas AK lesions adjacent to cSCCs display activated STAT3 in the nuclei. Our data suggest that miR-204 may act as a "rheostat" that controls the signalling towards the MAPK pathway or the STAT3 pathway in the progression from AK to cSCC.

Correction: Corrigendum: A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect

Nature Communications 5: Article number: 4608 (2014); Published: 3 April 2014; Updated: 2 November 2016 During the preparation of the PCR gel panels in Fig. 1b,c, duplicate or otherwise irrelevant lanes were excised; these excisions were not noted in the published figure as per the Nature journal policy.

Cancer pharmaco-epigenetics

Effects of <i>APOE</i>‐ε4 allele load on brain morphology in a cohort of middle‐aged healthy individuals with enriched genetic risk for Alzheimer's disease

Abstract Introduction Apolipoprotein E ( APOE )‐ε4 is the major genetic risk factor for Alzheimer's disease. However, the dose‐dependent impact of this allele on brain morphology of healthy individuals remains unclear. Methods We analyzed gray matter volumes (GMvs) in a sample of 533 healthy middle‐aged individuals with a substantial representation of ε4‐carriers (207 heterozygotes and 65 homozygotes). Results We found APOE ‐ε4 additive GMv reductions in the right hippocampus, caudate, precentral gyrus, and cerebellar crus. In these regions, the APOE genotype interacted with age, with homozygotes displaying lower GMv after the fifth decade of life. APOE ‐ε4 was also associated to greater GMv in the right thalamus, left occipital gyrus, and right frontal cortex. Discussion Our data indicate that APOE ‐ε4 exerts additive effects on GMv in regions relevant for Alzheimer's disease pathophysiology already in healthy individuals. These findings elucidate the mechanisms underlying the increased Alzheimer's disease risk in ε4‐carriers, suggesting a dose‐dependent disease vulnerability on the brain structure level.

IL-16 production is a mechanism of resistance to BTK inhibitors and R-CHOP in lymphomas

Introducing Bruton's tyrosine kinase (BTK) inhibitors has significantly improved outcomes for patients with B-cell malignancies and autoimmune disorders. However, resistance, either primary or acquired, remains a major clinical challenge. To better understand the underlying resistance mechanisms to BTK inhibitors, we established an ibrutinib-resistant model from a patient-derived splenic marginal zone lymphoma (MZL) cell line (VL51) through prolonged drug exposure. Resistant cells exhibited a 15-fold increase in ibrutinib's IC50, along with distinct morphological changes, mitochondrial activation, and cross-resistance to covalent, non-covalent BTK inhibitors and BTK degraders. Integrated transcriptomic, epigenomic, and proteomic analyses identified overexpression and secretion of IL-16 as a key feature of resistance, driven by chromatin remodeling and activation of the FLI1 transcription factor. IL-16 conferred ibrutinib resistance via CD9-mediated activation of the NF-kB and AKT signaling pathways and was found to be elevated in the serum of ibrutinib-refractory CLL patients. Functional studies showed that targeting the IL-16/CD9 axis using neutralizing antibodies or CD9-binding peptides restored sensitivity to BTK inhibitors and R-CHOP chemotherapy in MZL, mantle cell lymphoma, and diffuse large B-cell lymphoma models. These findings reveal a novel, targetable resistance mechanism with potential therapeutic implications for overcoming BTK inhibitor resistance in B-cell lymphomas.

Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia.

Glutathione S-transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents. The pi-class GST has been associated with preneoplastic and neoplastic changes. Recently, it has been reported that regulatory sequences near the GSTP1 gene, which encodes the human pi-class GST, are commonly hypermethylated in prostatic carcinomas. In the present study, we studied more than 300 primary human tumors originating in other organs for aberrant methylation of GSTP1 using methylation-specific PCR. GSTP1 hypermethylation was most frequent in breast and renal carcinoma, showing aberrant methylation in 30 and 20% of the cases, respectively. Other tumor types showed promoter methylation only rarely or not at all. Hypermethylation of GSTP1 was associated with loss of expression demonstrated by immunohistochemistry. Our results suggest that aberrant methylation of GSTP1 may contribute to the carcinogenetic process in breast and renal carcinomas.

FIGURE 5 from Single-cell Multiomics Analysis of Myelodysplastic Syndromes and Clinical Response to Hypomethylating Therapy

&lt;p&gt;Distribution of mutant cells within the BM compartments defined by the immunophenotype. &lt;b&gt;A,&lt;/b&gt; Percentage of mutant progenitor, immature erythroid and myeloid cells (Pro_Ery_Mye) compared with mutant lymphoid cells in each patient at diagnosis (top); percentage of mutant progenitor, immature erythroid and myeloid (bottom, left); or T, B, and NK cells (bottom, right) in each patient at diagnosis. &lt;b&gt;B,&lt;/b&gt; UMAP of BM cells from all samples colored by number of mutations per cell (left); cell type proportions according to the number of mutations per cell (right). &lt;b&gt;C,&lt;/b&gt; UMAP colored according to gene mutational status. WT, wild-type; MUT, mutant.&lt;/p&gt;

Local IL-17 orchestrates skin aging

We have found that increased local IL17 secreted by gamma/delta T cells, T helper 17 cells and ILCs, coordinate many of traits associated to skin aging, in particular those associated to increased inflammation. Importantly, inhibition of IL17 significantly ameliorates many of the aging related changes in the skin by boosting skin regeneration.

Figure S15 from ERBB4-Mediated Signaling Is a Mediator of Resistance to PI3K and BTK Inhibitors in B-cell Lymphoid Neoplasms

&lt;p&gt;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.&lt;/p&gt;

Epigenetic silencing of TGFβ1, BCL6, KILLIN and CTSZ is related to trastuzumab resistance in HER2-positive breast cancer models

FIGURE 6 from Single-cell Multiomics Analysis of Myelodysplastic Syndromes and Clinical Response to Hypomethylating Therapy

&lt;p&gt;Impact of AZA treatment on mutant cell populations in responder patients. &lt;b&gt;A,&lt;/b&gt; Mutant cell type proportions at diagnosis and after AZA treatment in responders. *, scCODA FDR &lt;0.1. &lt;b&gt;B,&lt;/b&gt; UMAPs at diagnosis and after AZA in all the HMA responder patients, colored by number of mutations per cell, highlight the three mutant populations significantly associated with response. &lt;b&gt;C,&lt;/b&gt; UMAPs highlighting mutant HSPCs in patient #2 (responder), mutant immature granulocytes in patient #9 (responder) and mutant immature monocytes in patient #7 (responder), colored by clone. Dx, diagnosis; Aza, after AZA treatment.&lt;/p&gt;

Arachidonic and Oleic Acid Exerts Distinct Effects on DNA Methylome

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.

Differences and Molecular Immunohistochemical Parameters in the Subtypes of Infiltrating Ductal Breast Cancer

Breast cancer is a heterogeneous disease, and patients are categorized into subtypes according to gene expression. We studied the associations among molecular, immunohistochemical, and clinicopathologic features and their distribution according to the subtypes luminal, HER2, basal, and normal-like in 60 patients with invasive ductal breast carcinoma without distant metastasis at the time of diagnosis (M0). We evaluated the hypermethylation of the CDH-1, RASSF1A, SIAH-1 and TSLC-1 genes by methylation-specific polymerase chain reaction and the expression of p53, bcl-2, cyclin D1, E-cadherin, and β-catenin proteins in tissue microarrays by immunohistochemical analysis. Expression of bcl-2 was associated with the luminal subtype (P = .003), and CDH-1 hypermethylation was present preferentially in HER2 tumors (P = .038). The basal subtype was characterized by the expression of β-catenin (P = .003). The hypermethylation of CDH-1 and the expression of bcl-2, cyclin D1, and β-catenin proteins differ among breast cancer subtypes.

Supplementary Table 1 from Genetic Unmasking of an Epigenetically Silenced microRNA in Human Cancer Cells

Supplementary Table 1 from Genetic Unmasking of an Epigenetically Silenced microRNA in Human Cancer Cells

An integrated genomic analysis of anaplastic meningioma identifies prognostic molecular signatures

Abstract Anaplastic meningioma is a rare and aggressive brain tumor characterised by intractable recurrences and dismal outcomes. Here, we present an integrated analysis of the whole genome, transcriptome and methylation profiles of primary and recurrent anaplastic meningioma. A key finding was the delineation of distinct molecular subgroups that were associated with diametrically opposed survival outcomes. Relative to lower grade meningiomas, anaplastic tumors harbored frequent driver mutations in SWI/SNF complex genes, which were confined to the poor prognosis subgroup. Aggressive disease was further characterised by transcriptional evidence of increased PRC2 activity, stemness and epithelial-to-mesenchymal transition. Our analyses discern biologically distinct variants of anaplastic meningioma with prognostic and therapeutic significance.