Xiaoying Chen

Linking DNA methylation and senescence in cancer cells: La méthylation de l'ADN est une modification épigénétique essentielle à la stabilité du génome, à la régulation de l'expression des gènes et à la répression des séquences répétées chez les mammifères. Après la réplication, les motifs de méthylation sont fidèlement reproduits par l'ADN méthyltransférase 1 (DNMT1) et son régulateur clé, UHRF1 (Ubiquitin-like with PHD and RING Finger Domains 1). UHRF1, grâce à ses multiples domaines fonctionnels, relie les modifications des histones à la méthylation de l'ADN et joue un rôle crucial dans le recrutement et l'activation de DNMT1 pour maintenir la méthylation. De plus, UHRF1 est fréquemment surexprimé dans les cancers et constitue un oncogène avéré in vivo. Cependant, il reste à déterminer si son action se limite à la régulation de DNMT1 ou si UHRF1 exerce d'autres fonctions importantes, notamment dans le contexte du cancer. Les schémas de méthylation aberrants de l'ADN sont une caractéristique commune des cancers humains. Étant donné que la méthylation de l'ADN est réversible, cibler cette machinerie représente une stratégie anti-tumorale prometteuse. Les inhibiteurs comme la 5-aza-déoxycytidine sont largement utilisés en recherche et en clinique, mais leur mécanisme d'action, souvent associé à des dommages à l'ADN, complique la distinction de leurs effets respectifs. Dans les lignées cellulaires de cancer colorectal humain HCT116 et DLD-1, nous avons utilisé un système degron inductible par auxine (AID) permettant une dégradation rapide et synchrone des protéines cibles. Nous avons observé que la déplétion d'UHRF1 entraîne une perte de méthylation de l'ADN bien plus importante que celle induite par l'absence de DNMT1. Cette hypométhylation n'est pas attribuable à une déméthylation passive, car les cellules dépourvues d'UHRF1 présentent des taux de prolifération réduits. Par des approches bioinformatiques, protéomiques et génétiques, nous avons également découvert qu'en plus d'activer DNMT1, UHRF1 interagit avec DNMT3A et DNMT3B, stimulant leurs activités, et qu'il antagonise la déméthylation active médiée par TET2. Ces résultats soulignent le rôle crucial d'UHRF1 dans l'homéostasie de la méthylation de l'ADN via plusieurs voies. Nous avons également exploré les effets de la déméthylation chronique de l'ADN dans les cellules cancéreuses. De manière surprenante, les cellules privées d'UHRF1 ou de DNMT1 ne meurent pas, mais entrent en sénescence. La déméthylation de l'ADN conduit à la surexpression de gènes impliqués dans la fonction lysosomale, le phénotype sécrétoire, la réponse à l'interféron et la voie p53. Toutefois, cette sénescence survient sans dommages à l'ADN et indépendamment des voies canoniques telles que p53 ou p16/pRB. Des mécanismes non canoniques, comme l'accumulation cytosolique de p21, favorisent les effets anti-apoptotiques. En outre, la suppression de la synthase GMP-AMP cyclique (cGAS) réduit le phénotype sécrétoire associé à la sénescence (SASP) et l'activité lysosomale, indépendamment de la voie STING. Des expériences de xénogreffes ont confirmé que la déméthylation chronique, notamment via la déplétion d'UHRF1, induit la sénescence in vivo. Mon travail met en lumière les rôles non canoniques d'UHRF1 et leurs implications pour des stratégies thérapeutiques avancées contre les cancers caractérisés par une méthylation aberrante. Nous avons montré que les cellules cancéreuses peuvent entrer en sénescence lorsque DNMT1 ou UHRF1 est inhibé, sans causer de dommages à l'ADN. Une thérapie combinée avec des sénolytiques pourrait ensuite cibler et éliminer ces cellules sénescentes, y compris dans des tumeurs dépourvues de p53, pRb ou p16 fonctionnels.

Inhibitors of DNA methylation such as 5-aza-deoxycytidine are widely used in experimental and clinical settings. However, their mechanism of action is such that DNA damage inevitably co-occurs with loss of DNA methylation, making it challenging to discern their respective effects. Here we deconvolute the effects of decreased DNA methylation and DNA damage on cancer cells, by using degron alleles of key DNA methylation regulators. We report that cancer cells with decreased DNA methylation—but no DNA damage—enter cellular senescence, with G1 arrest, SASP expression, and SA-β-gal positivity. This senescence is independent of p53 and Rb, but involves p21, which is cytoplasmic and inhibits apoptosis, and cGAS, playing a STING-independent role in the nucleus. Xenograft experiments show that tumor cells can be made senescent in vivo by decreasing DNA methylation. These findings reveal the intrinsic effects of loss of DNA methylation in cancer cells and have practical implications for future therapeutic approaches.

Inhibitors of DNA methylation such as 5-aza-deoxycytidine are widely used in experimental and clinical settings. However, their mechanism of action is such that DNA damage inevitably co-occurs with loss of DNA methylation, making it challenging to discern their respective effects. Here we deconvolute the effects of decreased DNA methylation and DNA damage on cancer cells, by using degron alleles of key DNA methylation regulators. We report that cancer cells with decreased DNA methylation —but no DNA damage— enter cellular senescence, with G1 arrest, SASP expression, and SA-β-gal positivity. This senescence is independent of p53 and Rb, but involves p21, which is cytoplasmic and inhibits apoptosis, and cGAS, playing a STING-independent role in the nucleus. Xenograft experiments show that tumor cells can be made senescent in vivo by decreasing DNA methylation. These findings reveal the intrinsic effects of loss of DNA methylation in cancer cells and have practical implications for future therapeutic approaches.

Epigenetic abnormalities are extremely widespread in cancer. Some of them are mere consequences of transformation, but some actively contribute to cancer initiation and progression; they provide powerful new biological markers, as well as new targets for therapies. In this review, we examine the recent literature and focus on one particular aspect of epigenome deregulation: large-scale chromatin changes, causing global changes of DNA methylation or histone modifications. After a brief overview of the one-dimension (1D) and three-dimension (3D) epigenome in healthy cells and of its homeostasis mechanisms, we use selected examples to describe how many different events (mutations, changes in metabolism, and infections) can cause profound changes to the epigenome and fuel cancer. We then present the consequences for therapies and briefly discuss the role of single-cell approaches for the future progress of the field.

Glycerol ester (GE) is a kind of important lipid in milk, which varies greatly depending on many factors. In this study, a novel pseudotargeted lipidomics strategy, named SWATH-MS2&1, was developed for the detection of GEs in milk and the Folch method was selected for the sample preparation. The developed method exhibited a competitive alternative to the acknowledged pseudotargeted strategy, including wider coverage (12 more GEs detected), higher repeatability (12 more GEs, whose coefficient of variation < 0.3), better linearity (5 more GEs, whose R² > 0.8), and similar sensitivity (only 2 GEs less than P-MRM after dilution). SWATH-MS2&1 was applied in the investigation of GEs from different milk samples. The orthogonal partial least-squares difference analysis of 219 GEs identified from SWATH-MS2&1 showed satisfying differentiation of different milk samples, and 76 GEs were screened out as potential markers. Our findings demonstrated that SWATH-MS2&1 could offer an accurate method to measure a wide spectrum of GEs in milk.

Mycoplasma hominis (MH) is an opportunistic pathogen, which often causes funisitis, spontaneous abortion, and low birth weight. However, current laboratory methods are time-consuming, labor-intensive, or require specialized laboratory instruments. Recombinase polymerase amplification (RPA) technology is a rapidly developing field because of the significance for clinical application and commercial value. Few studies have reported the use of RPA to detect MH. In this study, we developed the rapid MH detection assay, which may be potentially used as a sensitive point-of-care testing (POCT) in clinic. Primers based on the MH 16SrRNA gene and gap gene were explored and screened out. The probe of RPA-LFD was designed based on the optimal primer and confirmed. The reaction conditions of temperature and time for RPA were optimized. The sensitivity and specificity of the analysis were explored. A total of 60 clinical specimens were used to verify the efficiency of the two methods. The optimal reaction conditions were determined as 15 minutes and 39°C. The sensitivity of RPA was 10-6 ng for MH, which is 100,000 times more sensitive than traditional PCR. Moreover, we observed another six non-target reproductive tract common pathogens without amplification products. Furthermore, we found that there was no significant difference between RPA and the cultivation method (p > 0.05). These two methods were in good agreement (κ = 0.938) when detecting clinical specimens. A new method for sensitive and rapid detection of MH based on RPA was successfully developed, which can be applied in large-scale screening and as a supplementary method to classical methods.

There are disparities for the association between uncoupling proteins (UCP) and type 2 diabetes (T2DM). The study was to examine the associations of genetic variants of UCP2 and UCP3 with prediabetes and T2DM in a rural Chinese population. A population-based case-control study of 397 adults with T2DM, 394 with prediabetes and 409 with normal glucose tolerance (NGT) was carried out in 2014 in a rural community in eastern China. Three groups were identified through a community survey and the prediabetes and NGT groups were frequently matched by age and gender with the T2DM group and they were not relatives of T2DM subjects. With r2 ≥ 0.8 and minor allele frequency (MAF) ≥0.05 for tag single nucleotide polymorphisms (SNPs) with potential function, three (rs660339, rs45560234 and rs643064) and six (rs7930460, rs15763, rs647126, rs1800849, rs3781907 and rs1685356) SNPs were selected respectively for UCP2 and UCP3 and genotyped in real time using the MassARRAY system (Sequenom; USA). The haplotypes, gene-environmental interaction and association between genetic variants of UCP2 and UCP3 and prediabetes or T2DM were explored. There were no significant differences in age and sex among three study groups. After the adjustment for possible covariates, the A allele of rs1800849 in UCP3 was significantly associated with prediabetes (aORAA vs GG = 1.68, 95% CI: 1.02–2.78), and the association was also significant under the recessive model (aOR AA vs GA + GG = 1.64, 95% CI: 1.02–2.66). Also, rs15763 was found to be marginally significantly associated with T2DM under dominant model (ORGA + AA vs GG = 0.73, 95% CI: 0.52–1.03, P = 0.072). No haplotype was significantly associated with prediabetes or T2DM. Multiplicative interactions for rs660339-overweight on T2DM were observed. In addition, the AA genotype of rs660339 was associated with an increased risk of T2DM in overweight subjects (OR = 1.48, 95%CI: 0.87–2.52) but with a decreased risk in those with normal weight (OR = 0.54, 95%CI: 0.28–1.05). Rs1800849 in UCP3 was significantly associated with prediabetes. Overweight might modify the effects of rs660339 of UCP2 on T2DM.