Unveiling E2F4, TEAD1 and AP-1 as regulatory transcription factors of the replicative senescence program by multi-omics analysis

Senescence, a stable state of growth arrest, affects many physiological and pathophysiological processes, especially aging. Previous work has indicated that transcription factors (TFs) play a role in regulating senescence. However, a systematic study of regulatory TFs during replicative senescence (RS) using multi-omics analysis is still lacking. Here, we generated time-resolved RNA-seq, reduced representation bisulfite sequencing (RRBS) and ATAC-seq datasets during RS of mouse skin fibroblasts, which demonstrated that an enhanced inflammatory response and reduced proliferative capacity were the main characteristics of RS in both the transcriptome and epigenome. Through integrative analysis and genetic manipulations, we found that transcription factors E2F4, TEAD1 and AP-1 are key regulators of RS. Overexpression of E2f4 improved cellular proliferative capacity, attenuated SA-β-Gal activity and changed RS-associated differentially methylated sites (DMSs). Moreover, knockdown of Tead1 attenuated SA-β-Gal activity and partially altered the RS-associated transcriptome. In addition, knockdown of Atf3, one member of AP-1 superfamily TFs, reduced Cdkn2a (p16) expression in pre-senescent fibroblasts. Taken together, the results of this study identified transcription factors regulating the senescence program through multi-omics analysis, providing potential therapeutic targets for anti-aging.

nc886, a Non-Coding RNA, Is a New Biomarker and Epigenetic Mediator of Cellular Senescence in Fibroblasts

Functional studies of organisms and human models have revealed that epigenetic changes can significantly impact the process of aging. Non-coding RNA (ncRNA), one of epigenetic regulators, plays an important role in modifying the expression of mRNAs and their proteins. It can mediate the phenotype of cells. It has been reported that nc886 (=vtRNA2-1 or pre-miR-886), a long ncRNA, can suppress tumor formation and photo-damages of keratinocytes caused by UVB. The aim of this study was to determine the role of nc886 in replicative senescence of fibroblasts and determine whether substances capable of controlling nc886 expression could regulate cellular senescence. In replicative senescence fibroblasts, nc886 expression was decreased while methylated nc886 was increased. There were changes of senescence biomarkers including SA-β-gal activity and expression of p16INK4A and p21Waf1/Cip1 in senescent cells. These findings indicate that the decrease of nc886 associated with aging is related to cellular senescence of fibroblasts and that increasing nc886 expression has potential to suppress cellular senescence. AbsoluTea Concentrate 2.0 (ATC) increased nc886 expression and ameliorated cellular senescence of fibroblasts by inhibiting age-related biomarkers. These results indicate that nc886 has potential as a new target for anti-aging and that ATC can be a potent epigenetic anti-aging ingredient.

Senotherapeutic-like effect of Silybum marianum flower extract revealed on human skin cells

Cellular senescence causes irreversible growth arrest of cells. Prolonged accumulation of senescent cells in tissues leads to increased detrimental effects due to senescence associated secretory phenotype (SASP). Recent findings suggest that elimination of senescent cells has a beneficial effect on organismal aging and lifespan. In this study, using a validated replicative senescent human dermal fibroblasts (HDFs) model, we showed that elimination of senescent cells is possible through the activation of an apoptotic mechanism. We have shown in this replicative senescence model, that cell senescence is associated with DNA damage and cell cycle arrest (p21, p53 markers). We have shown that Silybum marianum flower extract (SMFE) is a safe and selective senolytic agent targeting only senescent cells. The elimination of the cells is induced through the activation of apoptotic pathway confirmed by annexin V/propidium iodide and caspase-3/PARP staining. Moreover, SMFE suppresses the expression of SASP factors such as IL-6 and MMP-1 in senescent HDFs. In a co-culture model of senescent and young fibroblasts, we demonstrated that senescent cells impaired the proliferative capacities of young cells. Interestingly, when the co-culture is treated with SMFE, the cell proliferation rate of young cells is increased due to the decrease of the senescent burden. Moreover, we demonstrated in vitro that senescent fibroblasts trigger senescent process in normal keratinocytes through a paracrine effect. Indeed, the conditioned medium of senescent HDFs treated with SMFE reduced the level of senescence-associated beta-galactosidase (SA-β-Gal), p16INK4A and SASP factors in keratinocytes compared with CM of senescent HDFs. These results indicate that SMFE can prevent premature aging due to senescence and even reprograms aged skin. Indeed, thanks to its senolytic and senomorphic properties SMFE is a candidate for anti-senescence strategies.

GH and Senescence: A New Understanding of Adult GH Action

Replicative senescence occurs due to an inability to repair DNA damage and activation of p53/p21 and p16INK4 pathways. It is considered a preventive mechanism for arresting proliferation of DNA-damaged cells. Stably senescent cells are characterized by a senescence-associated secretory phenotype (SASP), which produces and secretes cytokines, chemokines, and/or matrix metalloproteinases depending on the cell type. SASP proteins may increase cell proliferation, facilitating conversion of premalignant to malignant tumor cells, triggering DNA damage, and altering the tissue microenvironment. Further, senescent cells accumulate with age, thereby aggravating age-related tissue damage. Here, we review a heretofore unappreciated role for growth hormone (GH) as a SASP component, acting in an autocrine and paracrine fashion. In senescent cells, GH is activated by DNA-damage-induced p53 and inhibits phosphorylation of DNA repair proteins ATM, Chk2, p53, and H2AX. Somatotroph adenomas containing abundant intracellular GH exhibit increased somatic copy number alterations, indicative of DNA damage, and are associated with induced p53/p21. As this pathway restrains proliferation of DNA-damaged cells, these mechanisms may underlie the senescent phenotype and benign nature of slowly proliferating pituitary somatotroph adenomas. In highly proliferative cells, such as colon epithelial cells, GH induced in response to DNA damage suppresses p53, thereby triggering senescent cell proliferation. As senescent cells harbor unrepaired DNA damage, GH may enable senescent cells to evade senescence and reenter the cell cycle, resulting in acquisition of harmful mutations. These mechanisms, at least in part, may underlie pro-aging effects of GH observed in animal models and in patients with chronically elevated GH levels.

Dyskeratosis congenita: rare case report of Syria

ABSTRACT Dyskeratosis congenita (DC) is an inherited disease characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. Non-cutaneous abnormalities (dental, gastrointestinal, genitourinary, neurological, ophthalmic, pulmonary and skeletal) have also been reported. Bone marrow failure (BMF) is the main cause of early mortality, with an additional predisposition to malignancy. DC results from an anomalous progressive shortening of telomeres resulting in DNA replication problems inducing replicative senescence. Men are more affected than women are and X-linked recessive, autosomal dominant and autosomal recessive forms of the disease are recognized. There are no targeted therapies for DC. Patients treated with androgens had a hematological response. We herein describe case of a 32-year-old man, presented with several characteristic systemic features of this condition, including the classic triad of lesions, dysplastic bone marrow, epiphora and liver cirrhosis with grade I esophageal varices. Therefore, a prophylactic propranolol was started in additional to danazol. Three-week later, the patient had subsequent increases in his platelet, red cell and white cell counts.

Reviewing cancer’s biology: an eclectic approach

Background Cancer refers to a group of some of the worldwide most diagnosed and deadliest pathophysiological conditions that conquered researchers’ attention for decades and yet begs for more questions for a full comprehension of its complex cellular and molecular pathology. Main body The disease conditions are commonly characterized by unrestricted cell proliferation and dysfunctional replicative senescence pathways. In fact, the cell cycle operates under the rigorous control of complex signaling pathways involving cyclins and cyclin-dependent kinases assumed to be specific to each phase of the cycle. At each of these checkpoints, the cell is checked essentially for its DNA integrity. Genetic defects observed in these molecules (i.e., cyclins, cyclin-dependent kinases) are common features of cancer cells. Nevertheless, each cancer is different concerning its molecular and cellular etiology. These could range from the genetic defects mechanisms and/or the environmental conditions favoring epigenetically harbored homeostasis driving tumorigenesis alongside with the intratumoral heterogeneity with respect to the model that the tumor follows. Conclusions This review is not meant to be an exhaustive interpretation of carcinogenesis but to summarize some basic features of the molecular etiology of cancer and the intratumoral heterogeneity models that eventually bolster anticancer drug resistance for a more efficient design of drug targeting the pitfalls of the models.

Analysis of senescence-responsive stress fiber proteome reveals reorganization of stress fibers mediated by elongation factor eEF2 in HFF-1 cells

Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 135 proteins are associated with SFs, and 63 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

Pulmonary Alveolar Stem Cell Senescence, Apoptosis, and Differentiation by p53-Dependent and -Independent Mechanisms in Telomerase-Deficient Mice

Pulmonary premature ageing and fibrogenesis as in idiopathic pulmonary fibrosis (IPF) occur with the DNA damage response in lungs deficient of telomerase. The molecular mechanism mediating pulmonary alveolar cell fates remains to be investigated. The present study shows that naturally occurring ageing is associated with the DNA damage response (DDR) and activation of the p53 signalling pathway. Telomerase deficiency induced by telomerase RNA component (TERC) knockout (KO) accelerates not only replicative senescence but also altered differentiation and apoptosis of the pulmonary alveolar stem cells (AEC2) in association with increased innate immune natural killer (NK) cells in TERC KO mice. TERC KO results in increased senescence-associated heterochromatin foci (SAHF) marker HP1γ, p21, p16, and apoptosis-associated cleaved caspase-3 in AEC2. However, additional deficiency of the tumour suppressor p53 in the Trp53−/− allele of the late generation of TERC KO mice attenuates the increased senescent and apoptotic markers significantly. Moreover, p53 deficiency has no significant effect on the increased gene expression of T1α (a marker of terminal differentiated AEC1) in AEC2 of the late generation of TERC KO mice. These findings demonstrate that, in natural ageing or premature ageing accelerated by telomere shortening, pulmonary senescence and IPF develop with alveolar stem cell p53-dependent premature replicative senescence, apoptosis, and p53-independent differentiation, resulting in pulmonary senescence-associated low-grade inflammation (SALI). Our studies indicate a natural ageing-associated molecular mechanism of telomerase deficiency-induced telomere DDR and SALI in pulmonary ageing and IPF.

cGAS guards against chromosome end-to-end fusions during mitosis and facilitates replicative senescence

As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.

Senescence-Associated Secretory Phenotype as a Hinge Between Cardiovascular Diseases and Cancer

Overlapping risks for cancer and cardiovascular diseases (CVD), the two leading causes of mortality worldwide, suggest a shared biology between these diseases. The role of senescence in the development of cancer and CVD has been established. However, its role as the intersection between these diseases remains unclear. Senescence was originally characterized by an irreversible cell cycle arrest after a high number of divisions, namely replicative senescence (RS). However, it is becoming clear that senescence can also be instigated by cellular stress, so-called stress-induced premature senescence (SIPS). Telomere shortening is a hallmark of RS. The contribution of telomere DNA damage and subsequent DNA damage response/repair to SIPS has also been suggested. Although cellular senescence can mediate cell cycle arrest, senescent cells can also remain metabolically active and secrete cytokines, chemokines, growth factors, and reactive oxygen species (ROS), so-called senescence-associated secretory phenotype (SASP). The involvement of SASP in both cancer and CVD has been established. In patients with cancer or CVD, SASP is induced by various stressors including cancer treatments, pro-inflammatory cytokines, and ROS. Therefore, SASP can be the intersection between cancer and CVD. Importantly, the conventional concept of senescence as the mediator of cell cycle arrest has been challenged, as it was recently reported that chemotherapy-induced senescence can reprogram senescent cancer cells to acquire “stemness” (SAS: senescence-associated stemness). SAS allows senescent cancer cells to escape cell cycle arrest with strongly enhanced clonogenic growth capacity. SAS supports senescent cells to promote both cancer and CVD, particularly in highly stressful conditions such as cancer treatments, myocardial infarction, and heart failure. As therapeutic advances have increased overlapping risk factors for cancer and CVD, to further understand their interaction may provide better prevention, earlier detection, and safer treatment. Thus, it is critical to study the mechanisms by which these senescence pathways (SAS/SASP) are induced and regulated in both cancer and CVD.