Identification of intergenerational epigenetic inheritance by whole genome DNA methylation analysis in trios.

Genome-wide association studies have identified thousands of loci associated with common diseases and traits. However, a large fraction of heritability remains unexplained. Epigenetic modifications, such as the observed in DNA methylation have been proposed as a mechanism of intergenerational inheritance. To investigate the potential contribution of DNA methylation to the missing heritability, we analysed the methylomes of four healthy trios (two parents and one offspring) using whole genome bisulphite sequencing. Of the 1.5 million CpGs (19%) with over 20% variability between parents in at least one family and compatible with a Mendelian inheritance pattern, only 3488 CpGs (0.2%) lacked correlation with any SNP in the genome, marking them as potential sites for intergenerational epigenetic inheritance. These markers were distributed genome-wide, with some preference to be located in promoters. They displayed a bimodal distribution, being either fully methylated or unmethylated, and were often found at the boundaries of genomic regions with high/low GC content. This analysis provides a starting point for future investigations into the missing heritability of simple and complex traits.

Mycobacterium manresensis induces trained immunity in vitro.

The COVID-19 pandemic posed a global health crisis, with new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants weakening vaccine-driven protection. Trained immunity could help tackle COVID-19 disease. Our objective was to analyze whether heat-killed Mycobacterium manresensis (hkMm), an environmental mycobacterium, induces trained immunity and confers protection against SARS-CoV-2 infection. To this end, THP-1 cells and primary monocytes were trained with hkMm. The increased secretion of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1ß, and IL-10, metabolic activity, and changes in epigenetic marks suggested hkMm-induced trained immunity in vitro. Healthcare workers at risk of SARS-CoV-2 infection were enrolled into the MANRECOVID19 clinical trial (NCT04452773) and were administered Nyaditum resae (NR, containing hkMm) or placebo. No significant differences in monocyte inflammatory responses or the incidence of SARS-CoV-2 infection were found between the groups, although NR modified the profile of circulating immune cell populations. Our results show that M. manresensis induces trained immunity in vitro but not in vivo when orally administered as NR daily for 14 days.

Neuronal Replacement in Stem Cell Therapy for Stroke: Filling the Gap.

Stem cell therapy using human skin-derived neural precursors holds much promise for the treatment of stroke patients. Two main mechanisms have been proposed to give rise to the improved recovery in animal models of stroke after transplantation of these cells. First, the so called by-stander effect, which could modulate the environment during early phases after brain tissue damage, resulting in moderate improvements in the outcome of the insult. Second, the neuronal replacement and functional integration of grafted cells into the impaired brain circuitry, which will result in optimum long-term structural and functional repair. Recently developed sophisticated research tools like optogenetic control of neuronal activity and rabies virus monosynaptic tracing, among others, have made it possible to provide solid evidence about the functional integration of grafted cells and its contribution to improved recovery in animal models of brain damage. Moreover, previous clinical trials in patients with Parkinson's Disease represent a proof of principle that stem cell-based neuronal replacement could work in humans. Our studies with in vivo and ex vivo transplantation of human skin-derived cells neurons in animal model of stroke and organotypic cultures of adult human cortex, respectively, also support the hypothesis that human somatic cells reprogrammed into neurons can get integrated in the human lesioned neuronal circuitry. In the present short review, we summarized our data and recent studies from other groups supporting the above hypothesis and opening new avenues for development of the future clinical applications.

Novel Somatic Genetic Variants as Predictors of Resistance to EGFR-Targeted Therapies in Metastatic Colorectal Cancer Patients.

BACKGROUND: About 40% of RAS/BRAF wild-type metastatic colorectal cancer (mCRC) patients undergoing anti-EGFR-based therapy have poor outcomes. Treatment failure is not only associated with poorer prognosis but higher healthcare costs. Our aim was to identify novel somatic genetic variants in the primary tumor and assess their effect on anti-EGFR response. PATIENTS AND METHODS: Tumor (somatic) and blood (germline) DNA samples were obtained from two well-defined cohorts of mCRC patients, those sensitive and those resistant to EGFR blockade. Genetic variant screening of 43 EGFR-related genes was performed using targeted next-generation sequencing (NGS). Relevant clinical data were collected through chart review to assess genetic results. RESULTS: Among 61 patients, 38 were sensitive and 23 were resistant to treatment. We identified eight somatic variants that predicted non-response. Three were located in insulin-related genes (I668N and E1218K in IGF1R, T1156M in IRS2) and three in genes belonging to the LRIG family (T152T in LRIG1, S697L in LRIG2 and V812M in LRIG3). The remaining two variants were found in NRAS (G115Efs*46) and PDGFRA (T301T). We did not identify any somatic variants related to good response. CONCLUSIONS: This study provides evidence that novel somatic genetic variants along the EGFR-triggered pathway could modulate the response to anti-EGFR drugs in mCRC patients. It also highlights the influence of insulin-related genes and LRIG genes on anti-EGFR efficacy. Our findings could help characterize patients who are resistant to anti-EGFR blockade despite harboring RAS/BRAF wild-type tumors.

Tissue and cancer-specific expression of DIEXF is epigenetically mediated by an Alu repeat.

Alu repeats constitute a major fraction of human genome and for a small subset of them a role in gene regulation has been described. The number of studies focused on the functional characterization of particular Alu elements is very limited. Most Alu elements are DNA methylated and then assumed to lie in repressed chromatin domains. We hypothesize that Alu elements with low or variable DNA methylation are candidates for a functional role. In a genome-wide study in normal and cancer tissues, we pinpointed an Alu repeat (AluSq2) with differential methylation located upstream of the promoter region of the DIEXF gene. DIEXF encodes a highly conserved factor essential for the development of zebrafish digestive tract. To characterize the contribution of the Alu element to the regulation of DIEXF we analysed the epigenetic landscapes of the gene promoter and flanking regions in different cell types and cancers. Alternate epigenetic profiles (DNA methylation and histone modifications) of the AluSq2 element were associated with DIEXF transcript diversity as well as protein levels, while the epigenetic profile of the CpG island associated with the DIEXF promoter remained unchanged. These results suggest that AluSq2 might directly contribute to the regulation of DIEXF transcription and protein expression. Moreover, AluSq2 was DNA hypomethylated in different cancer types, pointing out its putative contribution to DIEXF alteration in cancer and its potential as tumoural biomarker.

Chainy: an universal tool for standardized relative quantification in real-time PCR.

Summary: Chainy is a cross-platform web tool providing systematic pipelines and steady criteria to process real-time PCR data, including the calculation of efficiencies from raw data by kinetic methods, evaluation of the suitability of multiple references, standardized normalization using one or more references, and group-wise relative quantification statistical testing. We illustrate the utility of Chainy for differential expression and chromatin immunoprecipitation enrichment (ChIP-QPCR) analysis. Availability and Implementation: Chainy is open source and freely available at http://maplab.cat/chainy. Contact: imallona@igtp.cat. Supplementary information: Supplementary data are available at Bioinformatics online.

The epigenetic landscape of Alu repeats delineates the structural and functional genomic architecture of colon cancer cells.

Cancer cells exhibit multiple epigenetic changes with prominent local DNA hypermethylation and widespread hypomethylation affecting large chromosomal domains. Epigenome studies often disregard the study of repeat elements owing to technical complexity and their undefined role in genome regulation. We have developed NSUMA (Next-generation Sequencing of UnMethylated Alu), a cost-effective approach allowing the unambiguous interrogation of DNA methylation in more than 130,000 individual Alu elements, the most abundant retrotransposon in the human genome. DNA methylation profiles of Alu repeats have been analyzed in colon cancers and normal tissues using NSUMA and whole-genome bisulfite sequencing. Normal cells show a low proportion of unmethylated Alu (1%-4%) that may increase up to 10-fold in cancer cells. In normal cells, unmethylated Alu elements tend to locate in the vicinity of functionally rich regions and display epigenetic features consistent with a direct impact on genome regulation. In cancer cells, Alu repeats are more resistant to hypomethylation than other retroelements. Genome segmentation based on high/low rates of Alu hypomethylation allows the identification of genomic compartments with differential genetic, epigenetic, and transcriptomic features. Alu hypomethylated regions show low transcriptional activity, late DNA replication, and its extent is associated with higher chromosomal instability. Our analysis demonstrates that Alu retroelements contribute to define the epigenetic landscape of normal and cancer cells and provides a unique resource on the epigenetic dynamics of a principal, but largely unexplored, component of the primate genome.

Expression of endoplasmic reticulum stress proteins is a candidate marker of brain metastasis in both ErbB-2+ and ErbB-2- primary breast tumors.

The increasing incidence of breast cancer brain metastasis in patients with otherwise well-controlled systemic cancer is a key challenge in cancer research. It is necessary to understand the properties of brain-tropic tumor cells to identify patients at risk for brain metastasis. Here we attempt to identify functional phenotypes that might enhance brain metastasis. To obtain an accurate classification of brain metastasis proteins, we mapped organ-specific brain metastasis gene expression signatures onto an experimental protein-protein interaction network based on brain metastatic cells. Thirty-seven proteins were differentially expressed between brain metastases and non-brain metastases. Analysis of metastatic tissues, the use of bioinformatic approaches, and the characterization of protein expression in tumors with or without metastasis identified candidate markers. A multivariate analysis based on stepwise logistic regression revealed GRP94, FN14, and inhibin as the best combination to discriminate between brain and non-brain metastases (ROC AUC = 0.85, 95% CI = 0.73 to 0.96 for the combination of the three proteins). These markers substantially improve the discrimination of brain metastasis compared with ErbB-2 alone (AUC = 0.76, 95% CI = 0.60 to 0.93). Furthermore, GRP94 was a better negative marker (LR = 0.16) than ErbB-2 (LR = 0.42). We conclude that, in breast carcinomas, certain proteins associated with the endoplasmic reticulum stress phenotype are candidate markers of brain metastasis.

Functional clustering of metastasis proteins describes plastic adaptation resources of breast-cancer cells to new microenvironments.

To examine the molecular mechanisms underlying breast cancer metastasis in liver and search for potential markers of metastatic progression in soft-tissue, we analyzed metastatic variants developed from the highly metastatic MDA-MB 435 cell line through in vivo stepwise selection in the athymic mice. Comparative proteomic analysis using two-dimensional electrophoresis (2DE-DIGE) revealed that 74 protein spots were reproducibly more than doubled in liver metastatic cells compared to parental counterpart. From 22 proteins identified by MALDI-TOF, belonging to intermediate filaments, intracellular transport and ATP synthesis, we generated a protein-protein interaction network containing 496 nodes, 12 of which interacted. GRP 75 was connected with four other proteins: prohibitin, HSP 27, elongin B and macropain delta chain. After functional classification, we found that pathways including hepatocyte growth factor receptor (p = 0.014), platelet-derived growth factor (p = 0.018), vascular endothelial growth factor (p = 0.021) and epidermal growth factor (p = 0.050) were predominant in liver metastatic cells, but not in lung metastatic cells. In conclusion, we suggest that GRP 75 is involved in cell proliferation, tumorigenesis and stress response in metastatic cells by recruiting signals in which the transmembrane receptor protein tyrosine kinase signaling pathway (p-value FDR = 1.71 x 10(-2)) and protein amino acid phosphorylation (p-value FDR = 3.28 x 10(-2)) might be the most significant biological process differentially increased in liver metastasis.

Biological pathways contributing to organ-specific phenotype of brain metastatic cells.

Secondary to the increased survival following chemotherapy, brain metastases have recently become a significant clinical problem for breast cancer patients. The aim of this study was to characterize those functional phenotypes that might enhance brain metastasis in breast cancer cells. We first analyzed by two-dimensional electrophoresis (2DE-DIGE) differences in protein expression between parental MDA-MB 435 cells and the brain metastatic variant 435-Br1, obtaining 19 identified proteins by peptide mass fingerprinting, 11 under-expressed (<2-fold) and 8 overexpressed (>2-fold) in 435-Br1. We created and analyzed protein interaction networks with a bioinformatic program (PIANA) from protein data, and it allowed us to associate 34/67-laminin receptor functionally with HSP 27, through a chaperone glucose-regulated protein GRP 94. Moreover, HSP 27 had the largest amount of direct and indirect protein interactions, forming a cluster of chaperones and cochaperones, associated through kinases to a set of intermediated filament proteins. In addition, functional groups of proteins identified were peptidase, DNA binding transcription factors, ATP synthase complex, anion transporters, and carbohydrate metabolism. Further functional analyses in cells, expression analyses in experimental tissues, and in human brain metastasis were addressed to validate the biological pathways contributing to organ-specific phenotype of brain metastasis.

Functional pathways shared by liver and lung metastases: a mitochondrial chaperone machine is up-regulated in soft-tissue breast cancer metastasis.

Genes that mediate breast cancer metastasis to lung are different from those which mediate bone metastasis. However, which markers accounts for the diversity of breast cancer metastasis remains unknown. The aim of this study was identify proteins associated with the soft-tissue metastatic ability of breast cancer tumors in metastases, coupling microarray data from clinical metastases and immunohistochemistry, for further screening for early detection at the first diagnosis in patients. We use a bioinformatic program to create and analyze protein interaction networks from protein experimental data, and to translate RNA expression analysis of breast cancer human metastases to protein, in a search for the phenotype associated with soft-tissue metastases. The pre-validated proteins constituted the protein signature for each metastasis: 37 (8.9%) from liver, 92 (8.5%) from lung and 167 (13%) from bone. Pleiotrophin, BAG 2, HSP 60 and vinculin were pre-validated in liver and lung metastases performing the soft-tissue phenotype. After IHC validation, we conclude that HSP 60, one of the best-known mitochondrial chaperone machines, is a key protein in soft-tissue metastases phenotype interacting with BAG 2, which competes for binding to GRP 75, the other mitochondrial chaperone. The relationship between HSP 60/GRP 75 and BAG 2 might result in the activation of several transcription pathways, different in liver from in lung metastases, as a nodal point coupling positive and negative actuators in the multiple survival-signal pathways and so achieving metastatic growth.

Underexpression of transcriptional regulators is common in metastatic breast cancer cells overexpressing Bcl-xL.

Bcl-xL gene induces metastasis in the lung, lymph nodes and bone when breast cancer cells are inoculated in Nude Balb/c mice. In an attempt to identify the molecules required for diverse metastatic foci, we compared gene expression levels in tumor cells and metastatic variants with a cDNA GeneFilter containing 4000 known genes. The transcriptional regulators of alpha1-fetoprotein transcription factor, TBP-associated factor 172 (TAF-172) and the human zinc finger protein 5 (ZFP5) were downregulated. The expression of TAF-172 was inversely proportional to Bcl-xL expression (ANOVA P < 0.0001) and metastatic activity (ANOVA P < 0.0001). A protein interaction program allowed us to functionally associate Bcl-xL and TAF through TATA-binding protein (TBP), suggesting that Bcl-xL connects metabolic pathways with transcriptional machinery. The prediction included proteins involved in apoptosis, electron transfer, kinases and transcription factors. These results indicate that the selection of diverse metastatic cells from the broad spectrum of tumor cell leads to the underexpression of certain transcriptional regulators that might act as adaptor molecules to different microenvironments, and indicate that the synergistic activity of several genes is needed for the selection process in several metastatic foci.

Bcl-x(L)-mediated changes in metabolic pathways of breast cancer cells: from survival in the blood stream to organ-specific metastasis.

Bcl-x(L) protein plays a role in breast cancer dormancy, promoting survival of cells in metastatic foci by counteracting the proapoptotic signals in the microenvironment. The aim of this study was to identify phenotypes mediated by Bcl-x(L) in breast cancer cells that enhance in vivo survival of clinical metastases. 435/Bcl-x(L) or 435/Neo human breast cancer cells were injected into the inguinal mammary gland of nude mice, and tumors, metastases in lymph node, lung, and bone, and bloodstream surviving cells were examined. Proteomic analysis identified 17 proteins that were overexpressed (more than twofold) or underexpressed (less than twofold) in metastases. A protein interaction program allowed us to functionally associate peroxiredoxin 3, peroxiredoxin 2, carbonyl reductase 3, and enolase 1, suggesting a role for cellular responses to oxidative stress in metastasis organ selection. The prediction included proteins involved in redox systems, kinase pathways, and the ATP synthase complex. Furthermore, the interaction of redox proteins with enolase 1 suggests a connection between glycolysis and antioxidant pathways, enabling achievement of a high metastatic activity. In conclusion, Bcl-x(L) mediates a phenotype in which redox pathways and glycolysis are coupled to protect breast cancer metastatic cells during transit from the primary tumor to the metastatic state.