Senescence-associated Secretory Phenotype Factors Research Articles

YAP/TAZ activation mediates PQ-induced lung fibrosis by sustaining senescent pulmonary epithelial cells.

Paraquat (PQ) is a widely used herbicide and a common cause of poisoning that leads to pulmonary fibrosis with a high mortality rate. However, the underlying mechanisms of PQ-induced pulmonary fibrosis and whether pulmonary epithelial cell senescence is involved in the process remain elusive. In this study, PQ-induced pulmonary epithelial cell senescence and Hippo-YAP/TAZ activation were observed in both C57BL/6 mice and human epithelial cells. PQ-induced senescent pulmonary epithelial cells promoted lung fibroblast transformation through secreting senescence-associated secretory phenotype (SASP) factors. Yap/Taz knockdown in mice lungs significantly decreased the expression of downstream profibrotic protein Ctgf and senescent markers p16 and p21, and alleviated PQ-induced pulmonary fibrosis. Interfering YAP/TAZ in senescent human pulmonary epithelial cells resulted in decreased expression of the anti-apoptosis protein survivin and elevated level of apoptosis. In conclusion, our findings reveal a novel mechanism by which the involvement of Hippo-YAP/TAZ activation in pulmonary epithelial cell senescence mediates the pathogenesis of PQ-induced pulmonary fibrosis, thereby offering novel insights and potential targets for the clinical management of PQ poisoning as well as providing the mechanistic insight of the involvement of Yap/Taz activation in cell senescence in pulmonary fibrosis and its related pulmonary disorders. The YIN YANG balance between cell senescence and apoptosis is important to maintain the homeostasis of the lung, the disruption of which will lead to disease.

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21+ vs p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

NEMO/NF-κB signaling functions as a double-edged sword in PanIN formation versus progression to pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is marked by a dismal survival rate, lacking effective therapeutics due to its aggressive growth, late-stage diagnosis, and chemotherapy resistance. Despite debates on NF-κB targeting for PDAC treatment, no successful approach has emerged.MethodsTo elucidate the role of NF-κB, we ablated NF-κB essential modulator (NEMO), critical for conventional NF-κB signaling, in the pancreata of mice that develop precancerous lesions (KC mouse model). Secretagogue-induced pancreatitis by cerulein injections was utilized to promote inflammation and accelerate PDAC development.ResultsNEMO deletion reduced fibrosis and inflammation in young KC mice, resulting in fewer pancreatic intraepithelial neoplasias (PanINs) at later stages. Paradoxically, however, NEMO deletion accelerated the progression of these fewer PanINs to PDAC and reduced median lifespan. Further, analysis of tissue microarrays from human PDAC sections highlighted the correlation between reduced NEMO expression in neoplastic cells and poorer prognosis, supporting our observation in mice. Mechanistically, NEMO deletion impeded oncogene-induced senescence (OIS), which is normally active in low-grade PanINs. This blockage resulted in fewer senescence-associated secretory phenotype (SASP) factors, reducing inflammation. However, blocked OIS fostered replication stress and DNA damage accumulation which accelerated PanIN progression to PDAC. Finally, treatment with the DNA damage-inducing reagent etoposide resulted in elevated cell death in NEMO-ablated PDAC cells compared to their NEMO-competent counterparts, indicative of a synthetic lethality paradigm.ConclusionsNEMO exhibited both oncogenic and tumor-suppressive properties during PDAC development. Caution is suggested in therapeutic interventions targeting NF-κB, which may be detrimental during PanIN progression but beneficial post-PDAC development.Graphical

Mechanistic role of RND3-regulated IL33/ST2 signaling on cardiomyocyte senescence

Hyperinflammatory responses are pivotal in the cardiomyocyte senescence pathophysiology, with IL33 serving as a crucial pro-inflammatory mediator. Our previous findings highlighted RND3's suppressive effect on IL33 expression. This study aims to explore the role of RND3 in IL33/ST2 signaling activation and in cardiomyocyte senescence. Intramyocardial injection of exogenous IL33 reduces the ejection fraction and fractional shortening of rats, inducing the appearance of senescence-associated secretory phenotype (SASP) in myocardial tissues. Recombinant IL33 treatment of AC16 cardiomyocytes significantly upregulated expression of SASP factors like IL1α, IL6, and MCP1, and increased the p-p65/p65 ratio and proportions of SA-β-gal and γH2AX-positive cells. NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) and ST2 antibody astegolimab treatments mitigated above effects. RND3 gene knockout H9C2 cardiomyocytes using CRISPR/Cas9 technology upregulated IL33, ST2L, IL1α, IL6, and MCP1 levels, decreased sST2 levels, and increased SA-β-gal and γH2AX-positive cells. A highly possibility of binding between RND3 and IL33 proteins was showed by molecular docking and co-immunoprecipitation, and loss of RND3 attenuated ubiquitination mediated degradation of IL33; what's more, a panel of ubiquitination regulatory genes closely related to RND3 were screened using qPCR array. In contrast, RND3 overexpression in rats by injection of AAV9-CMV-RND3 particles inhibited IL33, ST2L, IL1α, IL6, and MCP1 expression in cardiac tissues, decreased serum IL33 levels, and increased sST2 levels. These results suggest that RND3 expression in cardiomyocytes modulates cell senescence by inhibiting the IL33/ST2/NF-κB signaling pathway, underscoring its potential as a therapeutic target in cardiovascular senescence.

Analysis of the relationship between age-related erythrocyte dysfunction and fatigue.

With the declining birth rates and aging societies in developed countries, the average age of the working population is increasing. Older people tend to get tired more easily, so prevention of fatigue is important to improve the quality of life for older workers. This study aimed to assess the mechanism of fatigue in older people, especially focused on relation between dysfunction of erythrocyte and fatigue. Total power (TP), which is the value of autonomic nerve activity, was measured as a value of fatigue and significantly decreased in workers with aging. As properties of senescent erythrocytes, the erythrocyte sedimentation rate and damaged erythrocytes population increased with aging and correlated with TP. These results suggested that the accumulation of damaged erythrocytes contributes to fatigue. Recent studies revealed that senescence-associated secretory phenotype (SASP), a phenomenon in which senescent cells secrete a variety of cytokines, affected hematopoiesis in bone marrow. We analyzed the effects of SASP factors on erythropoiesis and found that Interleukin -1α (IL-1α) suppressed erythrocyte differentiation of hematopoietic stem cells in vitro. We also showed that IL-1α levels in human blood and saliva increase with aging, suggesting the possibility that IL-1α level in saliva can be used to predict the decline in hematopoietic function.

Delineating the heterogeneity of senescence-induced-functional alterations in hepatocytes

Background and aimCellular senescence of hepatocytes involves permanent cell cycle arrest, disrupted cellular bioenergetics, resistance to cell death, and the release of pro-inflammatory cytokines. This ‘zombie-like’ state perpetuates harmful effects on tissues and holds potential implications for liver disease progression. Remarkably, senescence exhibits heterogeneity, stemming from two crucial factors: the inducing stressor and the cell type. As such, our present study endeavors to characterize stressor-specific changes in senescence phenotype, its related molecular patterns, and cellular bioenergetics in primary mouse hepatocytes (PMH) and hepatocyte-derived liver organoids (HepOrgs).MethodsPMH, isolated by collagenase-perfused mouse liver (C57B6/J; 18–23 weeks), were cultured overnight in William’s E-medium supplemented with 2% FBS, l-glutamine, and hepatocyte growth supplements. HepOrgs were developed by culturing cells in a 3D matrix for two weeks. The senescence was induced by DNA damage (doxorubicin, cisplatin, and etoposide), oxidative stress (H2O2, and ethanol), and telomere inhibition (BIBR-1532), p53 activation (nutlin-3a), DNA methyl transferase inhibition (5-azacitidine), and metabolism inhibitors (galactosamine and hydroxyurea). SA-β galactosidase activity, immunofluorescence, immunoblotting, and senescence-associated secretory phenotype (SASP), and cellular bioenergetics were used to assess the senescence phenotype.ResultsEach senescence inducer triggers a unique combination of senescence markers in hepatocytes. All senescence inducers, except hydroxyurea and ethanol, increased SA-β galactosidase activity, the most commonly used marker for cellular senescence. Among the SASP factors, CCL2 and IL-10 were consistently upregulated, while Plasminogen activator inhibitor-1 exhibited global downregulation across all modes of senescence. Notably, DNA damage response was activated by DNA damage inducers. Cell cycle markers were most significantly reduced by doxorubicin, cisplatin, and galactosamine. Additionally, DNA damage-induced senescence shifted cellular bioenergetics capacity from glycolysis to oxidative phosphorylation. In HepOrgs exposed to senescence inducers, there was a notable increase in γH2A.X, p53, and p21 levels. Interestingly, while showing a similar trend, SASP gene expression in HepOrgs was significantly higher compared to PMH, demonstrating a several-fold increase.ConclusionIn our study, we demonstrated that each senescence inducer activates a unique combination of senescence markers in PMH. Doxorubicin demonstrated the highest efficacy in inducing senescence, followed by cisplatin and H2O2, with no impact on apoptosis. Each inducer prompted DNA damage response and mitochondrial dysfunction, independent of MAPK/AKT.Graphical

Cell type-dependent modulation of senescence features using Weo electrolyzed water.

Electrolyzed-reduced water has powerful antioxidant properties with constituents that scavenge reactive oxygen species (ROS), which are known to be produced by several intrinsic and extrinsic processes. When there is an imbalance between ROS production and antioxidant defenses, oxidative stress occurs. Persistent oxidative stress leads to cellular senescence, an important hallmark of aging, and is involved in several age-related conditions and illnesses. This study aims to investigate whether Weo electrolyzed water (WEW) could modulate the phenotype of senescent cells. We compared normal human lung fibroblasts (BJ) and breast cancer cells (T47D) treated with hydrogen peroxide (H2O2) to induce senescence. We assessed the molecular impact of WEW on markers of cellular senescence, senescence-associated secretory phenotype (SASP) factors, and stress response genes. Treatment with WEW modulated markers of cellular senescence, such as the senescence-associated β-galactosidase (SA-β-gal) activity, EdU incorporation and p21 expression, similarly in both cell types. However, WEW modulated the expression of SASP factors and stress response genes in a cell type-dependent and opposite fashion, significantly decreasing them in BJ cells, while stimulating their expression in T47D cells. Reduction in the expression of SASP factors and stress-related genes in BJ cells suggests that WEW acts as a protective factor, thereby reducing oxidative stress in normal cells, while making cancer cells more sensitive to the effects of cellular stress, thus increasing their elimination and consequently reducing their deleterious effects. These findings suggest that, due to its differential effects as a senomorphic factor, WEW could have a positive impact on longevity and age-related diseases.

Senolytic and senomorphic agent procyanidin C1 alleviates structural and functional decline in the aged retina

Increased cellular senescence burden contributes in part to age-related organ dysfunction and pathologies. In our study, using mouse models of natural aging, we observed structural and functional decline in the aged retina, which was accompanied by the accumulation of senescent cells and senescence-associated secretory phenotype factors. We further validated the senolytic and senomorphic properties of procyanidin C1 (PCC1) both in vitro and in vivo, the long-term treatment of which ameliorated age-related retinal impairment. Through high-throughput single-cell RNA sequencing (scRNA-seq), we comprehensively characterized the retinal landscape after PCC1 administration and deciphered the molecular basis underlying the senescence burden increment and elimination. By exploring the scRNA-seq database of age-related retinal disorders, we revealed the role of cellular senescence and the therapeutic potential of PCC1 in these pathologies. Overall, these results indicate the therapeutic effects of PCC1 on the aged retina and its potential use for treating age-related retinal disorders.

Celastrol mediates CAV1 to attenuate pro-tumorigenic effects of senescent cells

BackgroundCellular senescence is an emerging hallmark of cancers, primarily fuels cancer progression by expressing senescence-associated secretory phenotype (SASP). Caveolin-1 (CAV1) is a key mediator of cell senescence. Previous studies from our group have evidenced that the expression of CAV1 is downregulated by Celastrol (CeT). PurposeTo investigate the impact of CeT on cellular senescence and its subsequent influence on post-senescence-driven invasion, migration, and stemness of clear cell renal cell carcinoma (ccRCC). Study design and methodsThe expression levels of CAV1, canonical senescence markers, and markers associated with epithelial-mesenchymal transition (EMT) and stemness in clinical samples were assessed through Pearson correlation analysis. Senescent cell models were induced using DOX, and their impact on migration, invasion, and stemness was evaluated. The effects of CeT treatment on senescent cells and their pro-tumorigenic effects were examined. Subsequently, the underlying mechanism of CeT were explored using lentivirus transfection and CRISPR/Cas9 technology to silence CAV1. ResultsIn human ccRCC clinical samples, the expression of the canonical senescence markers p53, p21, and p16 are associated with ccRCC progression. Senescent cells facilitated migration, invasion, and enhanced stemness in both ccRCC cells and ccRCC tumor-bearing mice. As expected, CeT treatment reduced senescence markers (p16, p53, p21, SA-β-gal) and SASP factors (IL6, IL8, CXCL12), alleviating cell cycle arrest. However, it did not restore the proliferation of senescent cells. Additionally, CeT suppressed senescence-driven migration, invasion, and stemness. Further investigations into the underlying mechanism demonstrated that CAV1 is a critical mediator of cell senescence and represents a potential target for CeT to attenuate cellular senescence. ConclusionsThis study presents a pioneering investigation into the intricate interplay between cellular senescence and ccRCC progression. We unveil a novel mechanism of CeT to mitigate cellular senescence by downregulating CAV1, thereby inhibiting the migration, invasion and stemness of ccRCC driven by senescent cells. These findings provide valuable insights into the underlying mechanisms of CeT and its potential as a targeted therapeutic approach for alleviating the aggressive phenotypes associated with senescent cells in ccRCC.

High copper levels induce premature senescence in 3T3-L1 preadipocytes

Copper (Cu) dyshomeostasis has been linked to obesity and related morbidities and also to aging. Cu levels are higher in older or obese individuals, and adipose tissue (AT) Cu levels correlate with body mass index. Aging and obesity induce similar AT functional and structural changes, including an accumulation of senescent cells. To study the effect of Cu-mediated stress-induced premature senescent (Cu-SIPS) on preadipocytes, 3T3-L1 cell line was exposed to a subcytotoxic concentration of copper sulfate. After Cu treatment, preadipocytes acquired typical senescence characteristics including diminished cell proliferation, cell and nuclei enlargement and increased lysosomal mass (higher Lamp2 expression and a slight increased number of cells positive for β-galactosidase associated with senescence (SA-β-Gal)). Cell cycle arrest was due to upregulation of p16Ink4aInk4a and p21Waf1/Cip1. Accordingly, protein levels of the proliferation marker KI67 were reduced. Cu-SIPS relates with oxidative stress and, in this context, an increase of SOD1 and HO-1 expression was detected in Cu-treated cells. The mRNA expression of senescence-associated secretory phenotype factors, such as Mmp3, Il-6 and Tnf-α, increased in Cu-SIPS 3T3-L1 cells but no effect was observed on the expression of heterochromatin-associated protein 1(HP1).Although the downregulation of Lamin B1 expression is considered a hallmark of senescence, Cu-SIPS cells presented higher levels of Lamin B1. The dysregulation of nuclear lamina was accompanied by an increase of nuclear blebbing, but not of micronuclei number.To conclude, a Cu-SIPS model in 3T3-L1 preadipocytes is here described, which may be an asset to the study of AT dysregulation observed in obesity and aging.

Placental senescence pathophysiology is shared between peripartum cardiomyopathy and preeclampsia in mouse and human.

Peripartum cardiomyopathy (PPCM) is an idiopathic form of pregnancy-induced heart failure associated with preeclampsia. Circulating factors in late pregnancy are thought to contribute to both diseases, suggesting a common underlying pathophysiological process. However, what drives this process remains unclear. Using serum proteomics, we identified the senescence-associated secretory phenotype (SASP), a marker of cellular senescence associated with biological aging, as the most highly up-regulated pathway in young women with PPCM or preeclampsia. Placentas from women with preeclampsia displayed multiple markers of amplified senescence and tissue aging, as well as overall increased gene expression of 28 circulating proteins that contributed to SASP pathway enrichment in serum samples from patients with preeclampsia or PPCM. The most highly expressed placental SASP factor, activin A, was associated with cardiac dysfunction or heart failure severity in women with preeclampsia or PPCM. In a murine model of PPCM induced by cardiomyocyte-specific deletion of the gene encoding peroxisome proliferator-activated receptor γ coactivator-1α, inhibiting activin A signaling in the early postpartum period with a monoclonal antibody to the activin type II receptor improved heart function. In addition, attenuating placental senescence with the senolytic compound fisetin in late pregnancy improved cardiac function in these animals. These findings link senescence biology to cardiac dysfunction in pregnancy and help to elucidate the pathogenesis underlying cardiovascular diseases of pregnancy.

Abstract P46: Small Extracellular Vesicles from Therapy-induced Senescent TNBC Elicit Paracrine Anti-tumour Effects

Abstract Triple-negative breast cancers (TNBC), associated with poor prognosis and high tumour recurrence, are often treated with taxanes. However, cells may bypass treatment-induced cell death via mitotic slippage, resulting in multinucleated polyploid cells and therapy-induced senescence (TIS). TIS cancer cells represent a population of residual disease and may modulate therapy resistance and recurrence through secretion of the senescence-associated secretory phenotype (SASP). While SASP have been heavily linked with pro-tumorigenic behaviour mediated through soluble cytokines, small extracellular vesicles (sEVs) derived from TIS cancer cells represent an underappreciated aspect of the senescent tumour secretome, as its mechanistic role in mediating paracrine effects remains poorly understood. In this study, we uncovered an unexpected role for TIS-derived sEVs within the secretome, which conferred paracrine anti-tumour activity in recipient TNBC cancer cells, in contrast to SASP that support pro-tumorigenic behaviour. SEVs profiling through mass-spectrometry revealed that DKK1, a negative regulator of WNT signalling, is enriched in TIS-derived sEVs. Further investigation into the tumour microenvironment with animal models and neoadjuvant chemo-treated patient samples revealed STING-dependent immune response. Hence, our study suggests that sEVs should be considered as distinct SASP entities within the senescent tumour secretome and provide an additional basis for combinatorial use of senomorphics with taxanes to potentially modulate senescence-associated sEVs while suppressing pro-tumorigenic SASP factors. Citation Format: Matius Robert, Rekha Jakhar, Bijin Veonice Au, Gracie Wee Ling Eng, Alvin Kunyao Gao, You Heng Chuah, Karishma Sachaphibulkij, Lina Hsiu Kim Lim, Derrick Sek Tong Ong, Elaine Hsuen Lim, Yoko Itahana, Koji Itahana, Jit Kong Cheong, John Edward Connolly, Karen Carmelina Crasta. Small Extracellular Vesicles from Therapy-induced Senescent TNBC Elicit Paracrine Anti-tumour Effects [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P46.

Identification of Putative Causal Relationships between Blood-Based Biomarkers and Prediabetes-Induced Senescence: A Comprehensive Review

Prediabetes, a pivotal phase in glucose metabolism between normalcy and diabetes, exerts a profound influence on the aging process and the risk of age-related diseases. This comprehensive review delves into the intricate web of blood-based biomarkers that collectively expedite senescence, marking the transition from a state of health to age-related complications. Key findings underscore the significance of diverse biomarkers, such as telomere length, p16INK4a, senescence-associated secretory phenotype (SASP) factors, DNA methylation clocks, advanced glycation end products (AGEs), inflammatory and oxidative stress markers, circulating hormones, and additional factors such as folate, B12, and osteocalcin. Not only do these biomarkers serve as indicators of senescence but they also actively fuel chronic inflammation, oxidative stress, and metabolic dysregulation, all of which contribute to accelerated aging. The implications of this understanding are profound, as prediabetes emerges as a critical period in an individual’s life, influencing various physiological systems, including the vascular and neural systems, metabolic functions, hormonal regulation, and bone health. Recognizing the profound influence of prediabetes on senescence provides a foundation for personalized intervention strategies to mitigate age-related complications and promote healthy aging. Future research directions call for a more diverse array of biomarkers, the in-depth exploration of their roles, and the development of tailored precision medicine strategies to ensure a holistic understanding and effective management of prediabetes-induced senescence and its implications for aging. This knowledge has far-reaching implications for public health and clinical practice, emphasizing the need for early detection and intervention in prediabetic individuals to enhance the quality of life in an aging population with diverse needs.

Macrophage migration inhibitory factor mediates skin aging via CD74: Insights from single-cell and bulk RNA sequencing data

Cell-cell communication is crucial for regulating signaling and cellular function. However, the precise cellular and molecular changes remain poorly understood in skin aging. Based on single-cell and bulk RNA data, we explored the role of cell-cell ligand-receptor interaction in skin aging. We found that the macrophage migration inhibitory factor (MIF)/CD74 ligand-receptor complex was significantly upregulatedin aged skin, showing the predominant paracrine effect of keratinocytes on fibroblasts. Enrichment analysis and in vitro experiment revealed a close association of the activation of the MIF/CD74 with inflammatory pathways and immune response. Mechanistically, MIF/CD74 could significantly inhibit PPARγ protein, which thus significantly increased the degree of fibroblast senescence, and significantly up-regulated the expression of senescence-associated secretory phenotype (SASP) factors and FOS gene. Therefore, our study reveals that MIF/CD74 inhibits the activation of the PPAR signaling pathway, subsequently inducing the production of SASP factors and the upregulation of FOS expression, ultimately accelerating fibroblast senescence.

Mitochondrial injury induced by a Salmonella genotoxin triggers the proinflammatory senescence-associated secretory phenotype

Bacterial genotoxins damage host cells by targeting their chromosomal DNA. In the present study, we demonstrate that a genotoxin of Salmonella Typhi, typhoid toxin, triggers the senescence-associated secretory phenotype (SASP) by damaging mitochondrial DNA. The actions of typhoid toxin disrupt mitochondrial DNA integrity, leading to mitochondrial dysfunction and disturbance of redox homeostasis. Consequently, it facilitates the release of damaged mitochondrial DNA into the cytosol, activating type I interferon via the cGAS-STING pathway. We also reveal that the GCN2-mediated integrated stress response plays a role in the upregulation of inflammatory components depending on the STING signaling axis. These SASP factors can propagate the senescence effect on T cells, leading to senescence in these cells. These findings provide insights into how a bacterial genotoxin targets mitochondria to trigger a proinflammatory SASP, highlighting a potential therapeutic target for an anti-toxin intervention.

Kdm6a-CNN1 axis orchestrates epigenetic control of trauma-induced spinal cord microvascular endothelial cell senescence to balance neuroinflammation for improved neurological repair

Cellular senescence assumes pivotal roles in various diseases through the secretion of proinflammatory factors. Despite extensive investigations into vascular senescence associated with aging and degenerative diseases, the molecular mechanisms governing microvascular endothelial cell senescence induced by traumatic stress, particularly its involvement in senescence-induced inflammation, remain insufficiently elucidated. In this study, we present a comprehensive demonstration and characterization of microvascular endothelial cell senescence induced by spinal cord injury (SCI). Lysine demethylase 6A (Kdm6a), commonly known as UTX, emerges as a crucial regulator of cell senescence in injured spinal cord microvascular endothelial cells (SCMECs). Upregulation of UTX induces senescence in SCMECs, leading to an amplified release of proinflammatory factors, specifically the senescence-associated secretory phenotype (SASP) components, thereby modulating the inflammatory microenvironment. Conversely, the deletion of UTX in endothelial cells shields SCMECs against senescence, mitigates the release of proinflammatory SASP factors, and promotes neurological functional recovery after SCI. UTX forms an epigenetic regulatory axis by binding to calponin 1 (CNN1), orchestrating trauma-induced SCMECs senescence and SASP secretion, thereby influencing neuroinflammation and neurological functional repair. Furthermore, local delivery of a senolytic drug reduces senescent SCMECs and suppresses proinflammatory SASP secretion, reinstating a local regenerative microenvironment and enhancing functional repair after SCI. In conclusion, targeting the UTX-CNN1 epigenetic axis to prevent trauma-induced SCMECs senescence holds the potential to inhibit SASP secretion, alleviate neuroinflammation, and provide a novel treatment strategy for SCI repair.

Abstract 2954: Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT

Abstract Introduction: Androgen Deprivation Therapy (ADT) is a highly effective treatment for prostate cancer. However, resistance to ADT develops in most patients, leading to a lethal state of castration-resistant prostate cancer (CRPC). Senescent prostate cancer (SPC) cells can arise after ADT and contribute to therapy resistance. SPCs can restore replicative function and alter the tumor microenvironment through pro-tumorigenic senescence-associated secretory phenotype (SASP). We aimed to characterize changes in SASP in prostate cancer cell lines and serum samples from patients receiving ADT. Methods: SASP factors (Luminex 100/200 System, xMAP Instruments) were analyzed in patients undergoing ADT. To correlate SASP with the sensitivity to ADT, we sought to evaluate the regulation of SASP in vitro using PCa cell lines. The PC3 (AR resistant) and LNCaP (AR sensitive) cell lines were used in the experiments. We performed an analysis of archival tumor tissue (paired) from 18 patients with or without alterations in DNA repair genes (BRCA1, BRCA2, and CDK12). We performed immunohistochemical (IHC) staining of known SASP markers, such as P16, uPAR, gamma-H2AX, and Lamin B1. Results: In a cohort of patients (n=8) with pre and post-ADT matched status, we demonstrate increased levels of SASP factors such as IL-6 (pre-ADT: 1.54±0.24 pg/ml, post-ADT: 5.23±1.3 pg/ml p=0.001), IL-7 (pre-ADT: 3.11±0.6 pg/ml, post-ADT: 13.5±2.7 pg/ml p=0.01), IL-1 alpha (pre-ADT: 1.06±0.3 pg/ml, post-ADT: 15.3±3.5 pg/ml p=0.001), IL-15 (pre-ADT: 15.6±3.1 pg/ml, post-ADT: 37.1±4.8 pg/ml p=0.002), MIF (pre-ADT: 1036±67.6pg/ml, post-ADT: 2362±55.5 pg/ml p=0.003), and IL-15 (pre-ADT: 5±2.1 pg/ml, post-ADT: 37.5±5.1 pg/ml p=0.002). In vitro, the AR-responsive LNCaP cell line, exposed to AR blockade, showed similar findings in upregulating IL-6, IL-7, and IL-15 but not the PC3 cell line. We also demonstrate measurable differences between the expression of u-PAR, gamma-2AX, p16, and loss of Lamin B1 expression in tumor specimens from patients with advanced castration-resistant prostate cancer (n=18). The expression of p16 was significantly upregulated post-treatment irrespective of DDR status (p=0.0417). Other SASP factors showed a trend in upregulation but were insignificant (u-PAR p=0.056, gamma-H2AX p=0.62, Lamin B1 p=0.72) Conclusion: These results provide evidence of systemic increases in SASP-related cytokines following treatment with ADT. The in vitro results suggest that secretion of SASP is promoted by an AR-mediated mechanism. Preliminary results from tumor specimens suggest increased senescence marker p16 expression induced by therapy. Ongoing experiments are further characterizing the senescence markers in prostate tumor specimens and correlated to treatment effect. Citation Format: Maximilian Schwermann, Matthew Hadfield, Shaolei Lu, Praveen Srinivasan, Vida Tajiknia, William MacDonald, Andrew George, Shengliang Zhang, Leiqing Zhang, Kimberly Meza, Andre De Souza, Dragan Golijanin, Elias Hyams, Galina Lagos, Sheldon Holder, Anthony Mega, John Sedivy, Howard Safran, Wafik El-Deiry, Benedito Carneiro. Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2954.

Abstract 511: Cell cycle inhibitors exhibit anti-tumor immunomodulatory roles through the HIF-1-DDX41 cytosolic DNA sensing pathway in HCC

Abstract Cell cycle inhibitors have a long history as anti-cancer therapeutics in clinical applications. Uncontrolled and infinite cell cycles are hallmarks of cancer, which enable them to proliferate rapidly and survive. To target these key features of cancer cells, cell cycle inhibitors are used to arrest their active cell cycle and induce cell death. In this study, beyond the known primary effect on cell cycle control, we identified that an expected and a secondary effect in immune modulation by the cell cycle inhibitors. Anti-mitotic agents including Paclitaxel (microtubule stabilizer), Palbociclib (CDK4/6 inhibitor) and AZD1152 and GSK1070916 (Aurora Kinase B inhibitors) can also eliminate cancer cells via an alternative mechanism - the activation of STING signaling. Our results demonstrated that these anti-mitotic agents caused DNA damage and cytosolic DNA accumulation. The cytosolic DNA was captured by a cytosolic DNA sensor DDX41,which triggered STING-TBK1-IRF3/7 pathway to upregulate secretion of pro-inflammatory senescence-associated secretory phenotype (SASP) factors in cancer cells. In addition, we revealed that transcription of DDX41 was mediated by the transcription factor known ashypoxia-inducible factor (HIF). In HCC, hypoxia induced DDX41 expression through HIF-1,thus hypoxic HCC cells were more prone to STING activation and SASP production under mitotic stress induced by cell cycle inhibitors. In the tumor microenvironment, the SASP enhanced infiltration of immune cells into the tumor core to eliminate cancer cells. We also observed the additive effects of cell cycles inhibitors (Paclitaxel, Palbociclib, and AZD1152)with anti-PD-1 mAb to arrest growth of HCC in mouse models. In summary, this study exhibited the novel immune-mediated aspect of cell cycle inhibitors in suppressing tumor growth. Our data suggested the potential combination regimen of cell cycle inhibitors and currently available immunotherapy with promising result. Citation Format: Cerise Yuen Ki Chan, Po Yee Wong, Helen Do Gai Xue, Chi Ching Goh, Jacinth Wing Sum Cheu, Aki Pui Wah Tse, Misty Shuo Zhang, Carmen Chak Lui Wong. Cell cycle inhibitors exhibit anti-tumor immunomodulatory roles through the HIF-1-DDX41 cytosolic DNA sensing pathway in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 511.

Abstract 2964: Glutathione S-transferase omega 2 (GSTO2) suppresses the acquisition of the senescent tumor cells phenotype in lung adenocarcinoma

Abstract Cellular senescence is a phenomenon characterized by the cell cycle arrest and the induction of inflammatory responses, and is known to contribute to the development and progression of various diseases. In cancerous tissues, some cancer cells acquire the senescent traits, which are referred to as senescent tumor cells (STCs) and contribute to tumor invasion and metastasis. We have previously demonstrated that the expression of glutathione S-transferase omega 2 (GSTO2), which regulates the glutathione redox balance, is exclusively expressed in cells with regenerative ability such as airway basal cells, Clara cells, and type II alveolar cells in the lungs. Contrastingly, the expression of GSTO2 was completely lost in squamous cell carcinoma of the lung (Cancer Science, 2022); however, we found that some of lung adenocarcinoma expressed GSTO2. In this study, we aimed to clarify the significance of GSTO2 in lung adenocarcinoma, especially in the regulation of senescence and STCs. To examine whether GSTO2 expression affects senescence marker expression, we prepared GSTO2-transfected and mock-transfected lung adenocarcinoma cell line PC-9. Forced GSTO2 expression resulted in significantly reduced expression of senescence markers, such as p21, p53, and p16. We performed double staining for GSTO2 with p16 and confirmed the inverse correlation between them in lung adenocarcinoma tissues, where GSTO2-positive cells tended to express lower p16 level compared with GSTO2-negative cells. Furthermore, GSTO2-transfected PC-9 cells showed lower expression of senescence-associated secretory phenotype factors, such as IL-6, TNFα, and IL-1β than mock-transfected cells. These results suggest that GSTO2 suppresses senescence in lung adenocarcinoma cells. We next examined whether GSTO2 expression affects tumor cell growth because senescence is involved in the cell cycle arrest. Expectedly, MTT assay revealed that GSTO2 overexpression in PC-9 cells significantly promoted cell growth compared with the mock transfected cells. Contrastingly, the scratching assay showed that the cell migration of GSTO2-transfected PC-9 cells was significantly delayed compared with that of the mock-transfected cells. In conclusion, we demonstrated that GSTO2 regulates senescence, resulting in the suppression of cell migration. The expression of GSTO2 in lung adenocarcinoma may contribute to the inhibition of the emergence of STCs, being a cause of invasion and resistance to radiotherapy and chemotherapy. Citation Format: Hiroto Hatano, Ryusuke Sumiya, Teruki Hagiwara, Hiromu Suzuki, Kazuhiko Yamada, Norihiro Kokudo, Yuki Kawamura. Glutathione S-transferase omega 2 (GSTO2) suppresses the acquisition of the senescent tumor cells phenotype in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2964.

Identification of Osteoarthritis Inflamm-Aging Biomarkers by Integrating Bioinformatic Analysis and Machine Learning Strategies and the Clinical Validation

To identify inflamm-aging related biomarkers in osteoarthritis (OA). Microarray gene profiles of young and aging OA patients were obtained from the Gene Expression Omnibus (GEO) database and aging-related genes (ARGs) were obtained from the Human Aging Genome Resource (HAGR) database. The differentially expressed genes of young OA and older OA patients were screened and then intersected with ARGs to obtain the aging-related genes of OA. Enrichment analysis was performed to reveal the potential mechanisms of aging-related markers in OA. Three machine learning methods were used to identify core senescence markers of OA and the receiver operating characteristic (ROC) curve was used to assess their diagnostic performance. Peripheral blood mononuclear cells were collected from clinical OA patients to verify the expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. A total of 45 senescence-related markers were obtained, which were mainly involved in the regulation of cellular senescence, the cell cycle, inflammatory response, etc. Through the screening with the three machine learning methods, 5 core senescence biomarkers, including FOXO3, MCL1, SIRT3, STAG1, and S100A13, were obtained. A total of 20 cases of normal controls and 40 cases of OA patients, including 20 cases in the young patient group and 20 in the elderly patient group, were enrolled. Compared with those of the young patient group, C-reactive protein (CRP), interleukin (IL)-6, and IL-1β levels increased and IL-4 levels decreased in the elderly OA patient group (P<0.01); FOXO3, MCL1, and SIRT3 mRNA expression decreased and STAG1 and S100A13 mRNA expression increased (P<0.01). Pearson correlation analysis demonstrated that the selected markers were associated with some indicators, including erythrocyte sedimentation rate (ESR), IL-1β, IL-4, CRP, and IL-6. The area under the ROC curve of the 5 core aging genes was always greater than 0.8 and the C-index of the calibration curve in the nomogram prediction model was 0.755, which suggested the good calibration ability of the model. FOXO3, MCL1, SIRT3, STAG1, and S100A13 may serve as novel diagnostic biomolecular markers and potential therapeutic targets for OA inflamm-aging.