Changes in DNA methylation patterns in mammals with senescence, ageing and energy restriction

PhD ThesisDNA methylation is a reversible and inheritable chemical modification which involves the addition of a methyl group to DNA catalyzed by DNA methyltransferases (DNMTs), resulting in the conversion of the cytosine to 5’-methylcytosine, where the cytosine residue is followed by a guanine residue (CpG). In mammals, there are unusually dense clusters of CpG dinucleotides in the promoters of genes which are called “CpG islands”. For many housekeeping genes, these CpG islands are unmethylated in normal healthy tissue. However, CpG islands methylation is often associated with gene silencing and changed patterns of DNA methylation, associated with altered patterns of gene transcription, contribute to the aetiology of several diseases and to ageing. The overall aim of this project was to characterise the changes in DNA methylation which are observed during cell senescence (in the human cell line MRC-5) and during ageing and in response to dietary energy restriction (in various mice tissues). For this purpose: i) global DNA methylation was quantified using various techniques (ELISA, LINE-1, B1 and LUMA assays and HPLC); ii) site-specific genome-wide screening for methylation changes was performed using the MeDIP technique followed by hybridization to DNA microarrays; iii) validation of the results for selected candidates was performed by pyrosequencing and iv) investigation of mitochondrial DNA methylation was conducted using bisulphite-modified-DNA PCR direct sequencing. Effects of senescence on gene expression were assessed by transcriptome microarrays and by RT-qPCR studies. During the study, previously established methods for the investigation of global DNA methylation (LUMA) and site-specific methylation (MeDIP) were improved. Whilst global DNA methylation changes were detectable in senescence and after short-term dietary energy restriction, DNMTs/Dnmts expression changes were observed in senescence, ageing and following dietary energy restriction and were tissue- and treatment-specific. In parallel, site-specific aberrant DNA methylation was found in the promoters of the genes CTTN, GLIPR2, NPTX1 and SLC39A14 in replicative senescent MRC-5 human fibroblasts. These changes were validated by pyrosequencing and were accompanied by changes in expression of the corresponding genes. Also, in a pilot study, promoter methylation of several cell cycle genes was altered in replicative senescence with associated changes in gene expression. Concordant methylation changes were found in the promoters of 47 gene in ageing mouse and heart tissues including the genes Wnt5a, Map4k5, Apcdd1, Chp2 and Rasgrp2. In addition, dietary energy restriction counteracted the age-related DNA methylation changes in the promoters of 40 genes, including Aifm1, Irf8, Rarg, Nmi, Maf1, Rab33a and Fxn in mouse liver. Finally, mitochondrial DNA methylation studies revealed that senescence affected the DNA methylation patterns of the MT-COI and MT-ND1 gene coding sequences in MRC-5 fibroblasts whilst ageing affected the DNA methylation pattern of the D-Loop region in mouse liver, but this was not reversed by dietary energy restriction. Pathway analysis revealed that senescence- and age-related aberrant DNA methylation affected genes involved in inter-cellular communication, stress response, malignant transformation, cellular development/proliferation control, cell growth/differentiation and survival, apoptosis and immune response. As these genes contribute to the maintenance of cellular and tissue homeostasis, these findings suggest a potential role for altered DNA methylation in the aetiology of senescence and ageing. On the other hand, short-term dietary energy restriction modulated some of the age-related aberrant DNA methylation patterns of the ageing mouse liver, in particular those in promoters of genes involved in apoptosis regulation, inflammatory and immune response to viral infections, transcription regulation, vesicle trafficking and mitochondrial iron transport and respiration. Finally, mitochondrial DNA aberrant methylation - found to occur at genes belonging to Complex IV and to Complex I - may contribute to the accumulation of hazardous superoxide species in senescent cells whilst DNA aberrant methylation at the D-Loop mitochondrial regulatory region may contribute to age-related mitochondrial dysfunction. In conclusion, these findings suggest that altered DNA methylation may have a role in the aetiology of senescence and ageing and that some of the effects of dietary energy restriction in slowing down the ageing process and also delaying the onset of age-related diseases may occur via epigenetic mechanisms, including amelioration of age-related aberrations in patterns of DNA methylation

Low plasma PD-L1 levels, early tumor onset and absence of peritoneal carcinomatosis improve prognosis of women with advanced high-grade serous ovarian cancer

BackgroundThe most common subtype of ovarian cancer (OC) showing immunogenic potential is represented by the high-grade serous ovarian cancer (HGSOC), which is characterized by the presence of tumor-infiltrating immune cells able to modulate immune response. Because several studies showed a close correlation between OC patient's clinical outcome and expression of programmed cell death protein-1 or its ligand (PD-1/PD-L1), the aim of our study was to investigate if plasma levels of immunomodulatory proteins may predict prognosis of advanced HGSOC women.Patients and methodsThrough specific ELISA tests, we analyzed plasma concentrations of PD-L1, PD-1, butyrophilin sub-family 3A/CD277 receptor (BTN3A1), pan-BTN3As, butyrophilin sub-family 2 member A1 (BTN2A1), and B- and T-lymphocyte attenuator (BTLA) in one hundred patients affected by advanced HGSOC, before surgery and therapy. The Kaplan-Meier method was used to generate the survival curves, while univariate and multivariate analysis were performed using Cox proportional hazard regression models.ResultsFor each analyzed circulating biomarker, advanced HGSOC women were discriminated based on long (>= 30 months) versus short progression-free survival (PFS < 30 months). The concentration cut-offs, obtained by receiver operating characteristic (ROC) analysis, allowed to observe that poor clinical outcome and median PFS ranging between 6 and 16 months were associated with higher baseline levels of PD-L1 (> 0.42 ng/mL), PD-1 (> 2.48 ng/mL), BTN3A1 (> 4.75 ng/mL), pan-BTN3As (> 13.06 ng/mL), BTN2A1 (> 5.59 ng/mL) and BTLA (> 2.78 ng/mL). Furthermore, a lower median PFS was associated with peritoneal carcinomatosis, age at diagnosis > 60 years or Body Mass Index (BMI) > 25. A multivariate analysis also suggested that plasma concentrations of PD-L1 <= 0.42 ng/mL (HR: 2.23; 95% CI: 1.34 to 3.73; p = 0.002), age at diagnosis <= 60 years (HR: 1.70; 95% CI: 1.07 to 2.70; p = 0.024) and absence of peritoneal carcinomatosis (HR: 1.87; 95% CI: 1.23 to 2.85; p = 0.003) were significant prognostic marker for a longer PFS in advanced HGSOC patients.ConclusionsThe identification of high-risk HGSOC women could be improved through determination of the plasma PD-L1, PD-1, BTN3A1, pan-BTN3As, BTN2A1 and BTLA levels

Non-Small Cell Lung Cancer Harboring Concurrent EGFR Genomic Alterations: A Systematic Review and Critical Appraisal of the Double Dilemma

The molecular pathways which promote lung cancer cell features have been broadly explored, leading to significant improvement in prognostic and diagnostic strategies. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have dramatically altered the treatment approach for patients with metastatic non-small cell lung cancer (NSCLC). Latest investigations by using next-generation sequencing (NGS) have shown that other oncogenic driver mutations, believed mutually exclusive for decades, could coexist in EGFR-mutated NSCLC patients. However, the exact clinical and pathological role of concomitant genomic aberrations needs to be investigated. In this systematic review, we aimed to summarize the recent data on the oncogenic role of concurrent genomic alterations, by specifically evaluating the characteristics, the pathological significance, and their potential impact on the treatment approach

Inhibition of age-related therapy resistance in melanoma by rosiglitazone-mediated induction of Klotho

PURPOSE: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment, are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein, whose serum levels decrease dramatically by age 40. Studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma. EXPERIMENTAL DESIGN: PPARγ increases klotho levels, and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/ Wnt5A crosstalk, in vitro and in vivo, and the implications of that for targeted therapy in young vs. aged animals. RESULTS: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF-inhibitor sensitive, and BRAF inhibitor-resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors. CONCLUSIONS: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types

Deletion of Cyclophilin D Impairs β-Oxidation and Promotes Glucose Metabolism.

Cyclophilin D (CypD) is a mitochondrial matrix protein implicated in cell death, but a potential role in bioenergetics is not understood. Here, we show that loss or depletion of CypD in cell lines and mice induces defects in mitochondrial bioenergetics due to impaired fatty acid β-oxidation. In turn, CypD loss triggers a global compensatory shift towards glycolysis, with transcriptional upregulation of effectors of glucose metabolism, increased glucose consumption and higher ATP production. In vivo, the glycolytic shift secondary to CypD deletion is associated with expansion of insulin-producing β-cells, mild hyperinsulinemia, improved glucose tolerance, and resistance to high fat diet-induced liver damage and weight gain. Therefore, CypD is a novel regulator of mitochondrial bioenergetics, and unexpectedly controls glucose homeostasis, in vivo

DNA Damage-Induced Cell Cycle Regulation and Function of Novel Chk2 Phosphoresidues

Chk2 kinase is activated by DNA damage to regulate cell cycle arrest, DNA repair, and apoptosis. Phosphorylation of Chk2 in vivo by ataxia telangiectasia-mutated (ATM) on threonine 68 (T68) initiates a phosphorylation cascade that promotes the full activity of Chk2. We identified three serine residues (S19, S33, and S35) on Chk2 that became phosphorylated in vivo rapidly and exclusively in response to ionizing radiation (IR)-induced DNA double-strand breaks in an ATM- and Nbs1-dependent but ataxia telangiectasia- and Rad3-related-independent manner. Phosphorylation of these residues, restricted to the G(1) phase of the cell cycle, was induced by a higher dose of IR (>1 Gy) than that required for phosphorylation of T68 (0.25 Gy) and declined by 45 to 90 min, concomitant with a rise in Chk2 autophosphorylation. Compared to the wild-type form, Chk2 with alanine substitutions at S19, S33, and S35 (Chk2(S3A)) showed impaired dimerization, defective auto- and trans-phosphorylation activities, and reduced ability to promote degradation of Hdmx, a phosphorylation target of Chk2 and regulator of p53 activity. Besides, Chk2(S3A) failed to inhibit cell growth and, in response to IR, to arrest G(1)/S progression. These findings underscore the critical roles of S19, S33, and S35 and argue that these phosphoresidues may serve to fine-tune the ATM-dependent response of Chk2 to increasing amounts of DNA damage

Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues

<div><p>DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the “gold standard” of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.</p></div