Cellular Senescence: Defining a Path Forward.

Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers. We also present a resource tool to facilitate the identification of genes linked with senescence, SeneQuest (available at http://Senequest.net). Lastly, we propose an algorithm to accurately assess and quantify senescence, both in cultured cells and in vivo.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

A fluorophore-conjugated reagent enabling rapid detection, isolation and live tracking of senescent cells.

Cellular senescence is a stress-response mechanism implicated in various physiological processes, diseases, and aging. Current detection approaches have partially addressed the issue of senescent cell identification in clinical specimens. Effective methodologies enabling precise isolation or live tracking of senescent cells are still lacking. In-depth analysis of truly senescent cells is, therefore, an extremely challenging task. We report (1) the synthesis and validation of a fluorophore-conjugated, Sudan Black-B analog (GLF16), suitable for in vivo and in vitro analysis of senescence by fluorescence microscopy and flow cytometry and (2) the development and application of a GLF16-carrying micelle vector facilitating GLF16 uptake by living senescent cells in vivo and in vitro. The compound and the applied methodology render isolation of senescent cells an easy, rapid, and precise process. Straightforward nanocarrier-mediated GLF16 delivery in live senescent cells comprises a unique tool for characterization of senescence at an unprecedented depth.

Cellular senescence and cardiovascular diseases: moving to the "heart" of the problem.

Cardiovascular diseases (CVDs) constitute the prime cause of global mortality, with an immense impact on patient quality of life and disability. Clinical evidence has revealed a strong connection between cellular senescence and worse cardiac outcomes in the majority of CVDs concerning both ischemic and nonischemic cardiomyopathies. Cellular senescence is characterized by cell cycle arrest accompanied by alterations in several metabolic pathways, resulting in morphological and functional changes. Metabolic rewiring of senescent cells results in marked paracrine activity, through a unique secretome, often exerting deleterious effects on neighboring cells. Here, we recapitulate the hallmarks and key molecular pathways involved in cellular senescence in the cardiac context and summarize the different roles of senescence in the majority of CVDs. In the last few years, the possibility of eliminating senescent cells in various pathological conditions has been increasingly explored, giving rise to the field of senotherapeutics. Therefore, we additionally attempt to clarify the current state of this field with a focus on cardiac senescence and discuss the potential of implementing senolytics as a treatment option in heart disease.

Physiological hypoxia restrains the senescence-associated secretory phenotype via AMPK-mediated mTOR suppression.

Cellular senescence is a state of stable proliferative arrest triggered by damaging signals. Senescent cells persist during aging and promote age-related pathologies via the pro-inflammatory senescence-associated secretory phenotype (SASP), whose regulation depends on environmental factors. In vivo, a major environmental variable is oxygenation, which varies among and within tissues. Here, we demonstrate that senescent cells express lower levels of detrimental pro-inflammatory SASP factors in physiologically hypoxic environments, as measured in culture and in tissues. Mechanistically, exposure of senescent cells to low-oxygen conditions leads to AMPK activation and AMPK-mediated suppression of the mTOR-NF-κB signaling loop. Finally, we demonstrate that treatment with hypoxia-mimetic compounds reduces SASP in cells and tissues and improves strength in chemotherapy-treated and aged mice. Our findings highlight the importance of oxygen as a determinant for pro-inflammatory SASP expression and offer a potential new strategy to reduce detrimental paracrine effects of senescent cells.

A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization.

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.

Apidima Cave fossils provide earliest evidence of Homo sapiens in Eurasia.

Two fossilized human crania (Apidima 1 and Apidima 2) from Apidima Cave, southern Greece, were discovered in the late 1970s but have remained enigmatic owing to their incomplete nature, taphonomic distortion and lack of archaeological context and chronology. Here we virtually reconstruct both crania, provide detailed comparative descriptions and analyses, and date them using U-series radiometric methods. Apidima 2 dates to more than 170 thousand years ago and has a Neanderthal-like morphological pattern. By contrast, Apidima 1 dates to more than 210 thousand years ago and presents a mixture of modern human and primitive features. These results suggest that two late Middle Pleistocene human groups were present at this site-an early Homo sapiens population, followed by a Neanderthal population. Our findings support multiple dispersals of early modern humans out of Africa, and highlight the complex demographic processes that characterized Pleistocene human evolution and modern human presence in southeast Europe.

The survival of curaxins in the cancer arena.

With DNA damage being a primary anti-cancertarget, a need has arisen for the development of an approach that is a harmlessfor normal tissues but allows for cancer cell-specific cytotoxicity. Previous researchfrom K. Gurova's suggests that small compounds, namely curaxins that bind theDNA can cause chromatin instability and cell death in a cancer cell-specificmanner. In this brief perspective commentary, we investigate how the scientificcommunity has further developed this anti-cancer approach.

Loss of the tumour suppressor LKB1/STK11 uncovers a leptin-mediated sensitivity mechanism to mitochondrial uncouplers for targeted cancer therapy.

Non-small cell lung cancer (NSCLC) constitutes one of the deadliest and most common malignancies. The LKB1/STK11 tumour suppressor is mutated in ∼ 30% of NSCLCs, typically lung adenocarcinomas (LUAD). We implemented zebrafish and human lung organoids as synergistic platforms to pre-clinically screen for metabolic compounds selectively targeting LKB1-deficient tumours. Interestingly, two kinase inhibitors, Piceatannol and Tyrphostin 23, appeared to exert synthetic lethality with LKB1 mutations. Although LKB1 loss alone accelerates energy expenditure, unexpectedly we find that it additionally alters regulation of the key energy homeostasis maintenance player leptin (LEP), further increasing the energetic burden and exposing a vulnerable point; acquired sensitivity to the identified compounds. We show that compound treatment stabilises Hypoxia-inducible factor 1-alpha (HIF1A) by antagonising Von Hippel-Lindau (VHL)-mediated HIF1A ubiquitination, driving LEP hyperactivation. Importantly, we demonstrate that sensitivity to piceatannol/tyrphostin 23 epistatically relies on a HIF1A-LEP-Uncoupling Protein 2 (UCP2) signaling axis lowering cellular energy beyond survival, in already challenged LKB1-deficient cells. Thus, we uncover a pivotal metabolic vulnerability of LKB1-deficient tumours, which may be therapeutically exploited using our identified compounds as mitochondrial uncouplers.

Senescent cells in giant cell arteritis display an inflammatory phenotype participating in tissue injury via IL-6-dependent pathways.

OBJECTIVES: Age is the strongest risk factor of giant cell arteritis (GCA), implying a possible pathogenetic role of cellular senescence. To address this question, we applied an established senescence specific multimarker algorithm in temporal artery biopsies (TABs) of GCA patients. METHODS: 75(+) TABs from GCA patients, 22(-) TABs from polymyalgia rheumatica (PMR) patients and 10(-) TABs from non-GCA/non-PMR patients were retrospectively retrieved and analysed. Synovial tissue specimens from patients with inflammatory arthritis and aorta tissue were used as disease control samples. Senescent cells and their histological origin were identified with specific cellular markers; IL-6 and MMP-9 were investigated as components of the senescent associated secretory phenotype by triple costaining. GCA or PMR artery culture supernatants were applied to fibroblasts, HUVECs and monocytes with or without IL-6R blocking agent to explore the induction of IL-6-associated cellular senescence. RESULTS: Senescent cells were present in GCA arteries at higher proportion compared with PMR (9.50% vs 2.66%, respectively, p<0.0001) and were mainly originated from fibroblasts, macrophages and endothelial cells. IL-6 was expressed by senescent fibroblasts, and macrophages while MMP-9 by senescent fibroblasts only. IL-6(+) senescent cells were associated with the extension of vascular inflammation (transmural inflammation vs adventitia limited disease: 10.02% vs 4.37%, respectively, p<0.0001). GCA but not PMR artery culture supernatant could induce IL-6-associated senescence that was partially inhibited by IL-6R blockade. CONCLUSIONS: Senescent cells with inflammatory phenotype are present in GCA arteries and are associated with the tissue inflammatory bulk, suggesting a potential implication in disease pathogenesis.

53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling.

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a relatively greater impact on overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites. Recent progress in understanding how the rDNA damage response is organized has highlighted a key role of adaptor proteins. Here, we show that the scaffold tumor suppressor RASSF1A is recruited to rDNA breaks. RASSF1A recruitment to double-strand breaks is mediated by 53BP1 and depends on RASSF1A phosphorylation at Serine 131 by ATM kinase. Employing targeted rDNA damage, we uncover that RASSF1A recruitment promotes local ATM signaling. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Overall, we identify a novel role for RASSF1A at rDNA break sites, provide mechanistic insight into how the DNA damage response is organized in a chromatin context, and provide further evidence for how silencing of the RASSF1A tumor suppressor contributes to genome instability.

RASSF1A disrupts the NOTCH signaling axis via SNURF/RNF4-mediated ubiquitination of HES1.

RASSF1A promoter methylation has been correlated with tumor dedifferentiation and aggressive oncogenic behavior. Nevertheless, the underlying mechanism of RASSF1A-dependent tumor dedifferentiation remains elusive. Here, we show that RASSF1A directly uncouples the NOTCH-HES1 axis, a key suppressor of differentiation. Interestingly, the crosstalk of RASSF1A with HES1 occurs independently from the signaling route connecting RASSF1A with the Hippo pathway. At the molecular level, we demonstrate that RASSF1A acts as a scaffold essential for the SUMO-targeted E3 ligase SNURF/RNF4 to target HES1 for degradation. The reciprocal relationship between RASSF1A and HES1 is evident across a wide range of human tumors, highlighting the clinical significance of the identified pathway. We show that HES1 upregulation in a RASSF1A-depleted environment renders cells non-responsive to the downstream effects of γ-secretase inhibitors (GSIs) which restrict signaling at the level of the NOTCH receptor. Taken together, we report a mechanism through which RASSF1A exerts autonomous regulation of the critical Notch effector HES1, thus classifying RASSF1A expression as an integral determinant of the clinical effectiveness of Notch inhibitors.

Escape from senescence: molecular basis and therapeutic ramifications.

Cellular senescence constitutes a stress response mechanism in reaction to a plethora of stimuli. Senescent cells exhibit cell-cycle arrest and altered function. While cell-cycle withdrawal has been perceived as permanent, recent evidence in cancer research introduced the so-called escape-from-senescence concept. In particular, under certain conditions, senescent cells may resume proliferation, acquiring highly aggressive features. As such, they have been associated with tumour relapse, rendering senescence less effective in inhibiting cancer progression. Thus, conventional cancer treatments, incapable of eliminating senescence, may benefit if revisited to include senolytic agents. To this end, it is anticipated that the assessment of the senescence burden in everyday clinical material by pathologists will play a crucial role in the near future, laying the foundation for more personalised approaches. Here, we provide an overview of the investigations that introduced the escape-from-senescence phenomenon, the identified mechanisms, as well as the major implications for pathology and therapy. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage.

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.

Molecular biomarkers in cardio-oncology: Where we stand and where we are heading.

Until recently, cardiotoxicity in the setting of a malignant disease was attributed solely to the detrimental effects of chemo- and/or radio-therapy to the heart. On this account, the focus was on the evaluation of well-established cardiac biomarkers for the early detection of myocardial damage. Currently, this view has been revised. Cardiotoxicity is not restricted to a single organ but instead affects the endothelium as a whole. Indeed, it has come into light that not only cancer therapy but also malignant cells per se can impair the cardiovascular system, through a paracrine and endocrine mode of action. Even more intriguingly, a clear interplay between molecular pathways involved in cancer and cardiovascular disease has become prevalent, suggesting a common nominator that governs the pathophysiology of these two entities. Taken together, our strategy in the quest of novel biomarkers in the emerging field of cardio-oncology should be critically reshaped.

Helicobacter pylori Eradication Reverses DNA Damage Response Pathway but Not Senescence in Human Gastric Epithelium.

Helicobacter pylori (H. pylori) infection induces DNA Double-Strand Breaks (DSBs) and consequently activates the DNA Damage Response pathway (DDR) and senescence in gastric epithelium. We studied DDR activation and senescence before and after the eradication of the pathogen. Gastric antral and corpus biopsies of 61 patients with H. pylori infection, prior to and after eradication treatment, were analyzed by means of immunohistochemistry/immunofluorescence for DDR marker (γH2AΧ, phosporylated ataxia telangiectasia-mutated (pATM), p53-binding protein (53BP1) and p53) expression. Samples were also evaluated for Ki67 (proliferation index), cleaved caspase-3 (apoptotic index) and GL13 staining (cellular senescence). Ten H. pylori (-) dyspeptic patients served as controls. All patients were re-endoscoped in 72-1361 days (mean value 434 days), and tissue samples were processed in the same manner. The eradication of the microorganism, in human gastric mucosa, downregulates γH2AΧ expression in both the antrum and corpus (p = 0.00019 and p = 0.00081 respectively). The expression of pATM, p53 and 53BP1 is also reduced after eradication. Proliferation and apoptotic indices were reduced, albeit not significantly, after pathogen clearance. Moreover, cellular senescence is increased in H. pylori-infected mucosa and remains unaffected after eradication. Interestingly, senescence was statistically increased in areas of intestinal metaplasia (IM) compared with adjacent non-metaplastic mucosa (p < 0.001). In conclusion, H. pylori infection triggers DSBs, DDR and senescence in the gastric epithelium. Pathogen eradication reverses the DDR activation but not senescence. Increased senescent cells may favor IM persistence, thus potentially contributing to gastric carcinogenesis.

Pulsed Electromagnetic Fields (PEMFs) Trigger Cell Death and Senescence in Cancer Cells.

The currently available anti-cancer therapies, such as gamma-radiation and chemotherapeutic agents, induce cell death and cellular senescence not only in cancer cells but also in the adjacent normal tissue. New anti-tumor approaches focus on limiting the side effects on normal cells. In this frame, the potential anti-tumor properties of Pulsed Electromagnetic Fields (PEMFs) through the irradiation of breast cancer epithelial cells (MCF-7 and MDA-MB-231) and normal fibroblasts (FF95) were investigated. PEMFs had a frequency of 8 Hz, full-square wave type and magnetic flux density of 0.011 T and were applied twice daily for 5 days. The data collected showcase that PEMF application decreases the proliferation rate and viability of breast cancer cells while having the opposite effect on normal fibroblasts. Moreover, PEMF irradiation induces cell death and cellular senescence only in breast cancer cells without any effect in the non-cancerous cells. These findings suggest PEMF irradiation as a novel, non-invasive anti-cancer strategy that, when combined with senolytic drugs, may eliminate both cancer and the remaining senescent cells, while simultaneously avoiding the side effects of the current treatments.

Chronological Age and DNA Damage Accumulation in Blood Mononuclear Cells: A Linear Association in Healthy Humans after 50 Years of Age.

Aging is characterized by the progressive deregulation of homeostatic mechanisms causing the accumulation of macromolecular damage, including DNA damage, progressive decline in organ function and chronic diseases. Since several features of the aging phenotype are closely related to defects in the DNA damage response (DDR) network, we have herein investigated the relationship between chronological age and DDR signals in peripheral blood mononuclear cells (PBMCs) from healthy individuals. DDR-associated parameters, including endogenous DNA damage (single-strand breaks and double-strand breaks (DSBs) measured by the alkaline comet assay (Olive Tail Moment (OTM); DSBs-only by γH2AX immunofluorescence staining), DSBs repair capacity, oxidative stress, and apurinic/apyrimidinic sites were evaluated in PBMCs of 243 individuals aged 18-75 years, free of any major comorbidity. While OTM values showed marginal correlation with age until 50 years (rs = 0.41, p = 0.11), a linear relationship was observed after 50 years (r = 0.95, p < 0.001). Moreover, individuals older than 50 years showed increased endogenous DSBs levels (γH2Ax), higher oxidative stress, augmented apurinic/apyrimidinic sites and decreased DSBs repair capacity than those with age lower than 50 years (all p < 0.001). Results were reproduced when we examined men and women separately. Prospective studies confirming the value of DNA damage accumulation as a biomarker of aging, as well as the presence of a relevant agethreshold, are warranted.