State Of Growth Arrest Research Articles

Comprehensive assessment of cellular senescence in the tumor microenvironment.

Cellular senescence (CS), a state of permanent growth arrest, is intertwined with tumorigenesis. Due to the absence of specific markers, characterizing senescence levels and senescence-related phenotypes across cancer types remain unexplored. Here, we defined computational metrics of senescence levels as CS scores to delineate CS landscape across 33 cancer types and 29 normal tissues and explored CS-associated phenotypes by integrating multiplatform data from ~20 000 patients and ~212 000 single-cell profiles. CS scores showed cancer type-specific associations with genomic and immune characteristics and significantly predicted immunotherapy responses and patient prognosis in multiple cancers. Single-cell CS quantification revealed intra-tumor heterogeneity and activated immune microenvironment in senescent prostate cancer. Using machine learning algorithms, we identified three CS genes as potential prognostic predictors in prostate cancer and verified them by immunohistochemical assays in 72 patients. Our study provides a comprehensive framework for evaluating senescence levels and clinical relevance, gaining insights into CS roles in cancer- and senescence-related biomarker discovery.

CELLULAR SENESCENCE IMPAIRS CHONDROGENIC DIFFERENTIATION OF MESENCHYMAL STEM/STROMAL CELLS BY INHIBITING THE TRASFORMING GROWTH FACTOR-BETA SIGNALING

Purpose: Mesenchymal stem/stomal cells (MSCs) are often studied for their possible tissue Regenerative medicine. During in vitro expansion, however, MSCs may enter a state of permanent growth arrest - while remaining metabolically active - known as cellular senescence. Senescence can negatively affect tissue homeostasis and an increased amount of senescence cells can be found in pathological tissues, such as osteoarthritic cartilage. Although, it has been shown that cellular senescence may alters the differentiation capacity of MSCs towards the osteogenic and the adipogenic lineages, very little is known on how cellular senescence affect the chondrogenesis.

Role of senescent cells in the motile behavior of active, non-senescent cells in confluent populations

Characteristics of cell migration in a confluent population depend on the nature of cell-to-cell interactions as well as cell-intrinsic properties such as the directional persistence in crawling. In addition, biological tissues (or cell cultures) almost always carry anisotropies and they too can significantly affect cell motility. In the light of this viewpoint, the emergence of cellular senescences in a confluent population of active cells raises an interesting question. Cellular senescence is a process through which a cell enters a permanent growth-arrest state and generally exhibits a dramatic body expansion. Therefore, randomly emerging senescent cells transform an initially homogeneous cell population to a “binary mixture” of two distinct cell types. Here, using in vitro cultures of MDA-MB-231 cells we investigate how spatially localized cellular senescence affect the motility of active cells within a confluent population. Importantly, we estimate the intercellular surface energy of the interface between non-senescent and senescent MDA-MB-231 cells by combining the analysis on the motile behaviors of non-senescent cells encircling senescent cells and the result of extensive numerical simulations of a cellular Potts model. We find that the adhesion of normal cells to senescent cells is much weaker than that among normal cells and that the ‘arclength’ traveled by a normal cell along the boundary of a senescent cell, on average, is several times greater than the persistence length of normal cell in a densely packed homogeneous population. The directional persistent time of normal cell during its contact with a senescent cell also increases significantly. We speculate that the phenomenon could be a general feature associated with senescent cells as the enormous expansion of senescent cell’s membrane would inevitably decrease the density of cell adhesion molecules.

Why Senescent Cells Are Resistant to Apoptosis: An Insight for Senolytic Development

Cellular senescence is a process that leads to a state of irreversible cell growth arrest induced by a variety of intrinsic and extrinsic stresses. Senescent cells (SnCs) accumulate with age and have been implicated in various age-related diseases in part via expressing the senescence-associated secretory phenotype. Elimination of SnCs has the potential to delay aging, treat age-related diseases and extend healthspan. However, once cells becoming senescent, they are more resistant to apoptotic stimuli. Senolytics can selectively eliminate SnCs by targeting the SnC anti-apoptotic pathways (SCAPs). They have been developed as a novel pharmacological strategy to treat various age-related diseases. However, the heterogeneity of the SnCs indicates that SnCs depend on different proteins or pathways for their survival. Thus, a better understanding of the underlying mechanisms for apoptotic resistance of SnCs will provide new molecular targets for the development of cell-specific or broad-spectrum therapeutics to clear SnCs. In this review, we discussed the latest research progresses and challenge in senolytic development, described the significance of regulation of senescence and apoptosis in aging, and systematically summarized the SCAPs involved in the apoptotic resistance in SnCs.

Abstract P1-13-02: Autophagy inhibition and senolytics to improve the response to fulvestrant + palbociclib in ER positive breast cancer

Abstract While anti-estrogens or aromatase inhibitors in combination with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are the current standard of care for estrogen receptor-positive (ER+) Her-2 negative metastatic breast cancer, relapse after remission continues to be a serious limitation to the effectiveness of these therapies. Although these combination therapies prolong progression-free survival compared to endocrine therapy alone, the growth-arrested state of residual tumor cells is clearly transient. Tumor cells that escape what might be considered a dormant or quiescent state and regain proliferative capacity often acquire resistance to further therapies. Our studies are based upon the observation that breast tumor cells arrested by Fulvestrant + Palbociclib enter into a state of autophagy/senescence from which a subpopulation ultimately escapes, potentially contributing to disease recurrence. ER+ MCF-7 breast tumor cells that were induced into autophagy/senescence by Fulvestrant + Palbociclib in vitro were exposed to the autophagy inhibitors, chloroquine and bafilomycin, prior to as well as after growth arrest. However, autophagy inhibition only moderately enhanced the response to therapy and, at best, delayed proliferative recovery. These findings were confirmed by genetic inhibition of autophagy. Despite clear entry into a state of senescence, the Bcl-xl inhibitor and senolytic ABT-263 (navitoclax) failed to promote tumor cell death; this is likely a consequence of high levels of survivin in the MCF-7 breast tumor cell line. However, the BET inhibitor, ARV-825, eliminated a large fraction of the senescent cell population and significantly delayed proliferative recovery. Cell death did not appear to be a consequence of increased apoptosis. Additionally, analysis of three database sets demonstrated that higher breast cancer expression of BRD4, a primary target of ARV-825, correlated with lower recurrence-free survival compared to patients with cancers expressing lower levels of BRD4. These studies indicate that administration of BET inhibitors, which are currently being investigated in multiple clinical trials, may potentially improve standard of care therapy in metastatic ER+ breast cancer patients by prolonging progression-free survival. Citation Format: Ryan Michael Finnegan. Autophagy inhibition and senolytics to improve the response to fulvestrant + palbociclib in ER positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-13-02.

Aging, Cellular Senescence, and Alzheimer's Disease.

Aging is the greatest risk factor for late-onset Alzheimer’s disease (LOAD), which accounts for >95% of Alzheimer’s disease (AD) cases. The mechanism underlying the aging-related susceptibility to LOAD is unknown. Cellular senescence, a state of permanent cell growth arrest, is believed to contribute importantly to aging and aging-related diseases, including AD. Senescent astrocytes, microglia, endothelial cells, and neurons have been detected in the brain of AD patients and AD animal models. Removing senescent cells genetically or pharmacologically ameliorates β-amyloid (Aβ) peptide and tau-protein-induced neuropathologies, and improves memory in AD model mice, suggesting a pivotal role of cellular senescence in AD pathophysiology. Nonetheless, although accumulated evidence supports the role of cellular senescence in aging and AD, the mechanisms that promote cell senescence and how senescent cells contribute to AD neuropathophysiology remain largely unknown. This review summarizes recent advances in this field. We believe that the removal of senescent cells represents a promising approach toward the effective treatment of aging-related diseases, such as AD.

Tumor Dormancy: Biologic and Therapeutic Implications.

Metastatic cancer can arise years after treatment of the primary tumor because residual tumor cells can enter dormancy and evade elimination by anti-neoplastic therapies. The mechanisms underlying this phenomenon have been investigated and a number of hypotheses have been proposed. Tumor mass dormancy involves a balance between apoptotic and proliferative cells, keeping a micrometastatic lesion constant in size. This induces a need for blood supply which involves angiogenic dormancy. Cellular dormancy is also considered a mechanism of dormancy, where dormancy is induced due to cells entering a quiescent, reversible, growth-arrested state. In addition to all of these mechanisms, important changes in the tumor microenvironment, including the extracellular matrix, the oxygenation levels of the environment, and endoplasmic reticulum stress, are involved in inducing and maintaining tumor dormancy. Since dormant tumors are commonly known to be resistant to chemotherapy, gaining more knowledge of the mechanism of dormant tumor cells is of importance, as it can lead to the development of future treatment strategies.

MiRNA and lncRNA Expression Networks Modulate Cell Cycle and DNA Repair Inhibition in Senescent Prostate Cells

Cellular senescence is a state of permanent growth arrest that arises once cells reach the limit of their proliferative capacity. It creates an inflammatory microenvironment favouring the initiation and progression of various age-related diseases, including prostate cancer. Non-coding RNAs (ncRNAs) have emerged as important regulators of cellular gene expression. Nonetheless, very little is known about the interplay of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and how deregulation of ncRNA networks promotes cellular senescence. To investigate this, human prostate epithelial cells were cultured through different passages until senescent, and their RNA was extracted and sequenced using RNA sequencing (RNAseq) and microRNA sequencing (miRNA-seq) miRNAseq. Differential expression (DE) gene analysis was performed to compare senescent and proliferating cells with Limma, miRNA-target interactions with multiMiR, lncRNA-target interactions using TCGA data and network evaluation with miRmapper. We found that miR-335-3p, miR-543 and the lncRNAs H19 and SMIM10L2A all play central roles in the regulation of cell cycle and DNA repair processes. Expression of most genes belonging to these pathways were down-regulated by senescence. Using the concept of network centrality, we determined the top 10 miRNAs and lncRNAs, with miR-335-3p and H19 identified as the biggest hubs for miRNAs and lncRNA respectively. These ncRNAs regulate key genes belonging to pathways involved in cell senescence and prostate cancer demonstrating their central role in these processes and opening the possibility for their use as biomarkers or therapeutic targets to mitigate against prostate ageing and carcinogenesis.

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.

Pharmacological CDK4/6 inhibition reveals a p53‐dependent senescent state with restricted toxicity

Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti‐cancer drugs can cause premature senescence of non‐malignant cells. These therapy‐induced senescent cells can have pro‐tumorigenic and pro‐disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin‐dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence‐like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non‐malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i‐induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro‐inflammatory and NF‐κB‐driven components. We find that CDK4/6i‐induced senescent cells do not acquire pro‐tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress‐dependent and a predictor for the biological functions of different senescence subsets.

Oridonin inhibits SASP by blocking p38 and NF-κB pathways in senescent cells

Cellular senescence is a state of irreversible cell growth arrest that functions as a biological defense mechanism against severe DNA damage. Senescent cells with DNA damage produce pro-inflammatory cytokines, such as IL-6 and IL-8, and this phenomenon is called the senescence-associated secretory phenotype (SASP). SASP factors have been implicated in various disorders, including cancer. We performed a screening assay and identified oridonin as a candidate SASP inhibitor. Oridonin is an active diterpenoid that is isolated from Isodon plants and has been reported to exhibit anti-inflammatory, antibacterial, antioxidant, and antitumor activities. It reduced the secretion of IL-6 and IL-8 in senescent cells at the protein and mRNA levels. Oridonin also inhibited p65 subunit of NF-κB activity. However, oridonin did not affect SA β-gal activity and enhanced the expression of p21. The expression and phosphorylation of p38 were down-regulated by oridonin. The p38 inhibitor SB203580 inhibited the secretion of IL-8, slightly inhibited the secretion of IL-6, and did not affect NF-κB activity. Therefore, the NF-κB and p38 pathways may contribute to the inhibition of SASP by oridonin. Oridonin has potential as a therapeutic agent for SASP-related diseases.

Microenvironment-mediated cancer dormancy: Insights from metastability theory

Dormancy is an evolutionarily conserved protective mechanism widely observed in nature. A pathological example is found during cancer metastasis, where cancer cells disseminate from the primary tumor, home to secondary organs, and enter a growth-arrested state, which could last for decades. Recent studies have pointed toward the microenvironment being heavily involved in inducing, preserving, or ceasing this dormant state, with a strong focus on identifying specific molecular mechanisms and signaling pathways. Increasing evidence now suggests the existence of an interplay between intracellular as well as extracellular biochemical and mechanical cues in guiding such processes. Despite the inherent complexities associated with dormancy, proliferation, and growth of cancer cells and tumor tissues, viewing these phenomena from a physical perspective allows for a more global description, independent from many details of the systems. Building on the analogies between tissues and fluids and thermodynamic phase separation concepts, we classify a number of proposed mechanisms in terms of a thermodynamic metastability of the tumor with respect to growth. This can be governed by interaction with the microenvironment in the form of adherence (wetting) to a substrate or by mechanical confinement of the surrounding extracellular matrix. By drawing parallels with clinical and experimental data, we advance the notion that the local energy minima, or metastable states, emerging in the tissue droplet growth kinetics can be associated with a dormant state. Despite its simplicity, the provided framework captures several aspects associated with cancer dormancy and tumor growth.

The role of cellular senescence in female reproductive aging and the potential for senotherapeutic interventions.

BACKGROUNDAdvanced maternal age is associated with decreased oocyte quantity and quality as well as uterine and placental dysfunctions. These changes lead to infertility, pregnancy complications and birth defects in the offspring. As the mean age of giving birth is increasing worldwide, prevention of age-associated infertility and pregnancy complications, along with the more frequent use of ART, become extremely important. Currently, significant research is being conducted to unravel the mechanisms underlying female reproductive aging. Among the potential mechanisms involved, recent evidence has suggested a contributing role for cellular senescence, a cellular state of irreversible growth arrest characterized by a hypersecretory and pro-inflammatory phenotype. Elucidating the role of senescence in female reproductive aging holds the potential for developing novel and less invasive therapeutic measures to prevent or even reverse female reproductive aging and increase offspring wellbeing.OBJECTIVE AND RATIONALEThe review will summarize the positive and negative implications of cellular senescence in the pathophysiology of the female reproductive organs during aging and critically explore the use of novel senotherapeutics aiming to reverse and/or eliminate their detrimental effects. The focus will be on major senescence mechanisms of the ovaries, the uterus, and the placenta, as well as the potential and risks of using senotherapies that have been discovered in recent years.SEARCH METHODSData for this review were identified by searches of MEDLINE, PubMed and Google Scholar. References from relevant articles using the search terms ‘Cellular Senescence’, ‘Aging’, ‘Gestational age’, ‘Maternal Age’, ‘Anti-aging’, ‘Uterus’, ‘Pregnancy’, ‘Fertility’, ‘Infertility’, ‘Reproduction’, ‘Implant’, ‘Senolytic’, ‘Senostatic’, ‘Senotherapy’ and ‘Senotherapeutic’ where selected. A total of 182 articles published in English between 2005 and 2020 were included, 27 of which focus on potential senotherapies for reproductive aging. Exclusion criteria were inclusion of the terms ‘male’ and ‘plants’.OUTCOMESAging is a major determinant of reproductive wellbeing. Cellular senescence is a basic aging mechanism, which can be exploited for therapeutic interventions. Within the last decade, several new strategies for the development and repurposing of drugs targeting senescent cells have emerged, such as modulators of the anti-inflammatory response, oxidative stress, DNA damage, and mitochondria and protein dysfunctions. Several studies of female reproductive aging and senotherapies have been discussed that show promising results for future interventions.WIDER IMPLICATIONSIn most countries of the Organization for Economic Co-operation and Development, the average age at which women give birth is above 30 years. Currently, in countries such as the Netherlands, Australia, Spain, Finland, Germany and the UK, birth rates among 30- to 34-year-olds are now higher than in any other age groups. This review will provide new knowledge and scientific advancement on the senescence mechanisms during female reproductive aging, and benefit fundamental and clinical scientists and professionals in the areas of reproduction, cancer, immunobiology and fibrosis.

High dietary protein and fat contents exacerbate hepatic senescence and SASP in mice.

Dietary choices have a profound impact on the aging process. In addition to the total amount of energy intake, macronutrient composition influences both health and lifespan. However, the exact mechanisms by which dietary macronutrients influence onset and progression of age-associated features remain poorly understood. Cellular senescence is a state of stable growth arrest characterized by the secretion of numerous bioactive molecules with pro-inflammatory properties. Accumulation of senescent cells is considered one of the basic mechanisms of aging and an important contributor to chronic inflammation and tissue degeneration. Whether dietary macronutrients affect the accumulation and the phenotype of senescent cells with age is still unknown. Here, we show that feeding on diets with varying ratios of dietary macronutrients for 3 months has a significant effect on different senescence-associated markers in the mouse liver. High protein intake is associated with higher expression levels of the two classical senescence-associated growth arrest genes, p21 and p16. Furthermore, the expression of many pro-inflammatory secretory markers was increased in diets enriched in protein and further enhanced by increases in fat content. These results provide preliminary evidence that dietary macronutrients have a significant influence on senescence markers and merit further investigation.

In vitro antibacterial activity of nimbolide against Helicobacter pylori

Ethnopharmacological relevanceNimbolide is one of hundreds of phytochemicals that have been identified within the neem tree (Azadirachta indica A. Juss). As an evergreen tree native to the Indian subcontinent, components of the neem tree have been used for millennia in traditional medicine to treat dental, gastrointestinal, urinary tract, and blood-related ailments, ulcers, headaches, heartburn, and diabetes. In modern times, natural oils and extracts from the neem tree have been found to have activities against a variety of microorganisms, including human pathogens.Aim of the study: Helicobacter pylori, a prevalent gastric pathogen, shows increasing levels of antibiotic resistance. Thus, there is an increasing demand for novel therapeutics to treat chronic infections. The in vitro activity of neem oil extract against H. pylori was previously characterized and found to be bactericidal. Given the numerous phytochemicals found in neem oil extract, the present study was designed to define and characterize specific compounds showing bactericidal activity against H. pylori. Materials and methodsAzadirachtin, gedunin, and nimbolide, which are all common in neem extracts, were tested for antimicrobial activity; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for nine strains of H. pylori. The specific properties of nimbolide were further characterized against H. pylori strain G27. Bactericidal kinetics, reversibility, effectiveness at low pH, and activity under bacteriostatic conditions were examined. The hemolytic activity of nimbolide was also measured. Finally, neem oil extract and nimbolide effectiveness against H. pylori biofilms were examined in comparison to common antibiotics used to treat H. pylori infection. ResultsNimbolide, but not azadirachtin or gedunin, was effective against H. pylori; MICs and MBCs against the nine tested strains ranged between 1.25–5 μg/mL and 2.5–10 μg/mL, respectively. Additionally, neem oil extract and nimbolide were both effective against H. pylori biofilms. Nimbolide exhibited no significant hemolytic activity at biologically relevant concentrations. The bactericidal activity of nimbolide was time- and dose-dependent, independent of active H. pylori growth, and synergistic with low pH. Furthermore, nimbolide-mediated H. pylori cell death was irreversible after exposure to high nimbolide concentrations (80 μg/mL, after 2 h of exposure time and 40 μg/mL after 8 h of exposure). ConclusionsNimbolide has significant bactericidal activity against H. pylori, killing both free living bacterial cells as well as cells within a biofilm. Furthermore, the lack of hemolytic activity, synergistic activity at low pH and bactericidal properties even against bacteria in a state of growth arrest are all ideal pharmacological and biologically relevant properties for a potential new agent. This study underscores the potential of neem oil extract or nimbolide to be used as a future treatment for H. pylori infection.

Therapy-Induced Senescence As an Anti-Cancer and Immune-Stimulatory Strategy

Acute myeloid leukaemia (AML) is the most common type of leukaemia in elderly, for which the current gold standard of treatment is chemotherapy. Recently, it has been observed that AML blasts can activate the senescence program in response to chemotherapy (Therapy Induced Senescence, TIS). Cellular senescence is a stable and terminal state of growth arrest, often caused by nuclear DNA damage, associated with the transcriptional activation of a so-called Senescence Associated Secretory Phenotype (SASP), characterized, among others, by cytokines release, reported to promote immune-surveillance. Here we show that blasts, in response to chemotherapy, accumulate DNA damage and activate the senescence program, that in turn leads to HLA molecules upregulation, making them more prone to be cleared by T-cells.To evaluate TIS in AML blasts, we started by applying chemotherapy treatment (ARA-C) in six AML cell lines with different p53 status and FAB classification, observing reduction in proliferation rate and activation of DNA damage response pathways in the absence of overt apoptosis. We then quantified Senescence-Associated β-galactosidase (SA-βgal) activity and detected induction of senescence longitudinally with different extent. Accordingly, we observed the same features when applying ARA-C ex-vivo to primary AML samples collected at diagnosis.To delve deeper into the changes associated with the establishment of senescence in primary blasts, we performed RNA-seq analysis and observed an upregulation of pro-inflammatory genes (including IL1, IL6 and IL8) along with genes involved in immunogenicity. Investigating the biological significance of the transcriptional changes observed, we first reported an increase of HLA molecules on the surface of senescent blasts, as measured by FACS analyses. This observation prompted us to study the interaction between the immune system and senescent blasts, exploiting the Mixed Lymphocyte Reaction (MLR) assay. As expected, we detected a higher T-cell activation of both CD8+ and CD4+ subpopulations, accompanied by an increase in immunological synapses events and in apoptosis induction, when co-culturing chemotherapy treated blasts with T cells.In order to uncover the molecular mechanisms involved in TIS, we disentangled the role of DNA damage and cell cycle arrest in the phenotype observed comparing AML cell lines treatment with either ARA-C or the cdk4/6 inhibitor, which causes cell cycle arrest without inducing DNA damage. We found that HLA molecules overexpression is linked to the establishment of DNA damage response, however, when comparing acute to chronic ARA-C treatment, we observed that expression levels increased with treatment duration, suggesting that this feature is necessary but not sufficient to increase AML immunogenicity. Next, taking advantage of shRNAs (delivered by lentiviral vectors), we investigated deeper into the role of cell cylcle arrest. By stably knocking down p21, a crucial cell cycle inhibitor, we observed that ARA-C treated blasts had a reduced capacity of activating T-cell. Taken together, these observations point out to a crucial role for senescence in the improved immune-based clearance observed upon ARA-C treatment of blasts. Interestingly, a retrospective analysis showed that a cohort of patients clinically considered “responders” displayed a higher SA-βgal activity, further supporting the idea that senescence establishment in AML may act as a tumour suppressor mechanism.Overall, our study provides mechanistic insights into the biological and cellular response of AML cells to TIS and presents senescence as a positive mechanism able to promote AML eradication. This opens new lines of research aimed to develop novel therapeutic approaches against AML, exploiting senescence-induced features. DisclosuresVago: Moderna Therapeutics: Research Funding; GEN-DX: Patents & Royalties.

293 Dacarbazine affects the percentage of G0-positive melanoma cells

Cancer cell quiescence can be defined as reversible state of growth arrest in which cells enter to G0-phase of cell cycle. Dormant tumors characterized by the balance between apoptosis and cell proliferation rates resulting in the absence of the progression and clinical symptoms. Acquisition quiescence by cancer cells is flexible process which can be induced by various factors including chemotherapy. We aim to determine if chemotherapeutic agent dacarbazine (DTIC) may induce melanoma cell quiescence. Therefore the IC50 value of DTIC was calculated and the final concentration of DTIC was chosen which was defined as of low-to-moderate toxicity for cell lines tested. SK-MEL-2 cells showed two times increase of G0-positive cells whereas BRO cells exhibited decrease of G0-positive cells after DTIC treatment. Next, we performed a transcriptomic study in SK-MEL-2 cells and focused our attention on signaling pathways where the differentially expressed genes were also components. We observed the top downregulated genes to be components of the vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor R2 (VEGFR2), PI3K/AKT, cell cycle, p53 transcriptional gene network, DNA damage response, Focal Adhesion-PI3K-Akt-mTOR-signaling pathways. Besides, several signal pathways associated with altered microRNAs expression and functioning were found to be upregulated. We performed microRNA profiling and dysregulated microRNAs were identified. One of such microRNA, miR-204-5p was studied where gain-and-loss of function experiments showed it’s role in mitosis regulation under DTIC treatment. We suppose that DTIC may be used for melanoma cell quiescence modeling. The role of microRNAs as epigenetic regulators of tumor dormancy should be further evaluated.

Legacy Effect of Antioxidant N-acetylcysteine in Cellular Senescence of Diet-induced Obesity Mice.

Cellular senescence is a state of stable growth arrest triggered by mitogenic and metabolic stressors. Ageing and a high-fat diet (HFD) are proven inducers of senescence in various organs, presenting a challenge for ageing populations worldwide. Our previous study demonstrated that ROS scavenger N-acetylcysteine (NAC) can improve insulin resistance (IR) and chronic inflammation in diet-induced obesity mice, an effect better achieved through early intervention. We, herein, investigate whether NAC can improve cellular senescence in a diet-induced obesity mouse model, and whether a legacy effect is presented with early intervention. For a twelve-month treatment course, all C57B/L6 mice were fed a chow diet (CD), high-fat high-sucrose diet (HFD), CD+NAC1-12 (NAC intervention 1st-12th month), HFD+NAC1-12, and HFD+NAC1-6 (NAC intervention 1st-6th month). Staticalanalysis was used to analyze the different markers of cellular senescence and inflammation. Throughout the study, the HFD group exhibited significantly increased body weight (BW) and body fat, markers of senescence, decreased motor activity (MA) and impaired glucose tolerance. Compared to the HFD group, the HFD+NAC1-12 group exhibited increased MA, decreased BW and body fat, improved glucose tolerance, and decreased senescence markers.The HFD+NAC1-6 group showed similar effects to the HFD+NAC1-12 group, despite discontinuing NAC for 6 months. Our study showed that NAC significantly increased MA in both HFD+NAC1-12 and HFD+NAC1-6 groups, and improved HFD-induced mitochondrial and intracellular ROS expression, DNA and protein oxidative damage, and adipose tissue inflammation. Legacy effect was indeed presented in HFD-induced cellular senescence with NAC intervention, with possible mechanisms being persistently increased motor activity and anti-oxidative stress effects.

Abstract 2025: Characterization of cancer cell lines made senescent by exposure to ribociclib, doxorubicin, or TGFβ1, and identification of genes required for entry into senescence and senescent cell survival

Abstract Cellular senescence is a stress-induced state of stable growth arrest characterized by high expression of cell cycle inhibitors; a dramatic change in cell morphology, including an increase in lysosomal content; and secretion of large numbers of proteins involved in immune signaling and extracellular matrix remodeling. The physiological importance of cellular senescence has been attributed to prevention of carcinogenesis, aging, development, and tissue repair, and tumor cells can undergo senescence in response to therapeutic agents. In this work, we sought to validate the senescence-inducing activity of two known inducers (doxorubicin and TGFβ1) and a CDK4/6 inhibitor (ribociclib) and to identify proteins that can kill senescent tumor cells (senolytic targets) if knocked out and to identify downstream components of the tumor cell senescence pathway. Huh7 hepatocellular carcinoma cells and SK-MEL-28 melanoma cells were induced to senescence by treatment with three different agents: ribociclib, low doses of doxorubicin, or TGFβ1. The induction of senescence was confirmed by observing growth arrest, an increase in SA-β-gal staining, dramatic cell morphology changes, loss of c-Myc protein, increased expression of p15, and increased expression of senescence-associated secretory phenotype (SASP) proteins. Induction of SASP components was measured by RNAseq and SOMAscan. All three agents induced a senescent state, with blockage at different stages of the cell cycle observed. Induction of known immune factors, including IL-8 and IL-11, were identified in senescent cells (Huh7). A whole-genome CRISPR screen identified proteins required to enter senescence and those that were incompatible with the senescent state if knocked out. Expected hits were observed (eg, TGFBR1/TGFBR2 for TGFβ1, RB for ribociclib, and TOP2A for doxorubicin) for guide DNAs (gDNAs) that blocked entry into the senescent state. No gDNA candidates for common downstream senescence pathway components were observed, suggesting that these components are essential genes or that they do not exist. gDNA-induced knockouts that were incompatible with the senescent state dropped out of the screen and represent potential senolytic targets. The screen identified BCL2L1 as the only common senolytic hit across multiple senescence-inducing reagents, confirming published reports suggesting it is a senolytic target. These data show that ribociclib, doxorubicin, and TGFβ1 induced senescence in cancer cell lines. Whole-genome CRISPR screens identified senescence pathway components for each of these agents, as well as a common senolytic target. Citation Format: Pasupuleti Rao, Jennifer Tullai, Peter Aspesi, Felipa Mapa, Nadire Cochran, Frederic Sigoillot, Guglielmo Roma, Scott Gleim, Jaison Jacob, Jason Marchese, Jonathan Solomon. Characterization of cancer cell lines made senescent by exposure to ribociclib, doxorubicin, or TGFβ1, and identification of genes required for entry into senescence and senescent cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2025.

Reduction of lamin B receptor levels by miR-340-5p disrupts chromatin, promotes cell senescence and enhances senolysis.

A major stress response influenced by microRNAs (miRNAs) is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR-340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding lamin B receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains, promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for removing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in pathologies of human aging.