Senescence-associated Secretory Phenotype Genes Research Articles

Glutaminase-1 inhibition alleviates senescence of Wharton's jelly-derived mesenchymal stem cells via senolysis.

Replicative senescence of mesenchymal stem cells (MSCs) caused by repeated cell culture undermines their potential as a cell therapy because of the reduction in their proliferation and therapeutic potential. Glutaminase-1 (GLS1) is reported to be involved in the survival of senescent cells, and inhibition of GLS1 alleviates age-related dysfunction via senescent cell removal. In the present study, we attempted to elucidate the association between MSC senescence and GLS1. We conducted in vitro and in vivo experiments to analyze the effect of GLS1 inhibition on senolysis and the therapeutic effects of MSCs. Inhibition of GLS1 in Wharton's jelly-derived MSCs (WJ-MSCs) reduced the expression of aging-related markers, such as p16, p21, and senescence-associated secretory phenotype genes, by senolysis. Replicative senescence-alleviated WJ-MSCs, which recovered after short-term treatment with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES), showed increased proliferation and therapeutic effects compared to those observed with senescent WJ-MSCs. Moreover, compared to senescent WJ-MSCs, replicative senescence-alleviated WJ-MSCs inhibited apoptosis in serum-starved C2C12 cells, enhanced muscle formation, and hindered apoptosis and fibrosis in mdx mice. These results imply that GLS1 inhibition can ameliorate the therapeutic effects of senescent WJ-MSCs in patients with muscle diseases such as Duchenne muscular dystrophy. In conclusion, GLS1 is a key factor in modulating the senescence mechanism of MSCs, and regulation of GLS1 may enhance the therapeutic effects of senescent MSCs, thereby increasing the success rate of clinical trials involving MSCs.

Radix rehmanniae praeparata extracts ameliorate hepatic ischemia-reperfusion injury by reversing LRP1-NOTCH1-C/EBPβ axis-mediated senescence fate of LSECs

BackgroundHepatic ischemia-reperfusion injury (HIRI) is commonly observed in cases of extensive hepatic resection and involves complex mechanisms. Cell senescence has been recognized as a factor in liver injury including HIRI, where it presents as a pro-inflammatory phenotype called senescence-associated secretory phenotype (SASP). Radix Rehmanniae Praeparata (RRP) is a commonly utilized traditional Chinese medicine known for its hepatoprotective, anti-aging and antioxidant qualities. Despite its recognized benefits, the specific mechanisms by which RRP may impede the progression of HIRI through the regulation of cell senescence and the identification of the most potent anti-aging extracts from RRP remain unclear. Materials and methodsHere, we first applied different chemical analysis methods to identify the RRP aqueous extract (RRPAE) and active fractions of RRP. Next, we constructed a surgically established mouse model and a hypoxia-reoxygenation (HR)-stimulated liver sinusoidal endothelial cells (LSECs) model to explore the underlying mechanism of RRP against HIRI through transcriptomics and multiple molecular biology experiments. ResultsAfter identifying active ingredients in RRP, we observed that RRP and its factions effectively restored LSECs fenestration and improved inflammation, cellular swelling and vascular continuity in the hepatic sinusoidal region during HIRI. Transcriptomic results revealed that RRP might reverse HIRI-induced senescence through the NOTCH signaling pathway and cell categorization further showed that the senescent cell population in HIRI liver was primarily LSECs rather than other cell types. Different RRPAE, especially RRP glucoside (RRPGLY), improved LSECs senescence and suppressed the expression of pro-inflammatory SASP genes either induced by HR insult or NOTCH1 activator, which was accompanied with the inhibition of LRP1-NOTCH1-C/EBPβ pathways. Additionally, the specific inhibition of NOTCH1 by siRNA synergistically enhanced the hepatoprotective effect of RRPGLY. The ChIP-qPCR results further showed that C/EBPβ was enriched at the promoter of a representative SASP, Il-1β, in hypoxic LSECs but was significantly inhibited by RRPGLY. ConclusionOur study not only clarified the potential mechanism of RRP active extractions in alleviating HIRI, but also highlighted RRPGLY was the main component of RRP that exerted anti-aging and anti-HIRI effects, providing a fresh perspective on the use of RRP to improve HIRI.

Senomorphic activity of a combination of niacinamide and hyaluronic acid: correlation with clinical improvement of skin aging

Intrinsic and extrinsic factors, including lifestyle and sun exposure, can contribute to cell senescence, which impairs skin homeostasis, that may in turn lead to skin aging. Senescent cells have a specific secretome, called the senescence-associated secretory phenotype (SASP) that includes MMPs, CXCLs and S100A8/9. Reducing the SASP with senotherapeutics is a promising strategy to reduce skin aging. Here we evaluated the effect of a formula containing niacinamide and hyaluronic acid, which are known to limit senescence and skin aging. We conducted three different studies. (1) Ex vivo explants treated with the formula had more collagen and glycosaminoglycan. (2) In a clinical trial with forty-four women, two months of treatment improved fine lines, wrinkles, luminosity, smoothness, homogeneity, and plumpness. (3) In a third study on thirty women, we treated one arm for two months and took skin biopsies to study gene expression. 101 mRNAs and 13 miRNAs were differentially expressed. We observed a likely senomorphic effect, as there was a decrease in many SASP genes including MMP12 and CXCL9 and a significant downregulation of autocrine signaling genes: S100A8 and S100A9. These pharmaco-clinical results are the first to demonstrate the senomorphic properties of an effective anti-aging formula in skin.

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

Exploring Transcriptional Regulation of Beta Cell SASP by Brd4-Associated Proteins and Cell Cycle Control Protein p21.

Type 1 diabetes (T1D) is a metabolic disease resulting from progressive autoimmune destruction of insulin-producing pancreatic beta cells. Although the majority of beta cells are lost in T1D, a small subset undergoes senescence, a stress response involving growth arrest, DNA damage response, and activation of a senescence-associated secretory phenotype (SASP). SASP in beta cells of the nonobese diabetic (NOD) mouse model of T1D and primary human islets is regulated at the level of transcription by bromodomain extra-terminal (BET) proteins, but the mechanisms remain unclear. To explore how SASP is transcriptionally regulated in beta cells, we used the NOD beta cell line NIT-1 to model beta cell SASP and identified binding partners of BET protein Brd4 and explored the role of the cyclin-dependent kinase inhibitor p21. Brd4 interacted with a variety of proteins in senescent NIT-1 cells including subunits of the Ino80 chromatin remodeling complex, which was expressed in beta cells during T1D progression in NOD mice and in human beta cells of control, autoantibody-positive, and T1D donors as determined from single-cell RNA-seq data. RNAi knockdown of p21 during senescence in NIT-1 cells did not significantly impact viability or SASP. Taken together, these results suggest that Brd4 interacts with several protein partners during senescence in NIT-1 cells, some of which may play roles in SASP gene activation and that p21 is dispensable for the SASP in this beta cell model.

Abstract A035: Radiation-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting matrix metalloproteinases, chemokines, cytokines, and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells as well as promoted tumor cell proliferation and radioresistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and publicly available data from the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells, like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also observed in PDX models of GBM. Using these PDX models, we are currently validating novel senolytic-based therapeutic approaches to mitigate tumor recurrence after radiotherapy. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Ben R. Jordan, Nozomi Tomimatsu, Luis Macedo Di Cristofaro, Suman Kanji, Bipasha Mukherjee, Sandeep Burma. Radiation-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A035.

Abstract A009: DNA damage-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and the normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting proteases, cytokines and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these cells senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells and promoted tumor cell proliferation and radiation resistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also seen in multiple PDX models of GBM. Using these PDX lines, we demonstrate that adjuvant senolytic treatment can delay or even prevent recurrence in pre-clinical mouse models of GBM. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Sandeep Burma, Nozomi Tomimatsu, Luis Cristofaro, Suman Kanji, Benjamin Jordan, Bipasha Mukherjee. DNA damage-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A009.

Abstract PR001: DNA damage-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and the normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting proteases, cytokines and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these cells senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells and promoted tumor cell proliferation and radiation resistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also seen in multiple PDX models of GBM. Using these PDX lines, we demonstrate that adjuvant senolytic treatment can delay or even prevent recurrence in pre-clinical mouse models of GBM. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Sandeep Burma, Nozomi Tomimatsu, Luis Cristofaro, Suman Kanji, Benjamin Jordan, Bipasha Mukherjee. DNA damage-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR001.

YBX1 Regulates Satellite II RNA Loading into Small Extracellular Vesicles and Promotes the Senescent Phenotype.

Senescent cells secrete inflammatory proteins and small extracellular vesicles (sEVs), collectively termed senescence-associated secretory phenotype (SASP), and promote age-related diseases. Epigenetic alteration in senescent cells induces the expression of satellite II (SATII) RNA, non-coding RNA transcribed from pericentromeric repetitive sequences in the genome, leading to the expression of inflammatory SASP genes. SATII RNA is contained in sEVs and functions as an SASP factor in recipient cells. However, the molecular mechanism of SATII RNA loading into sEVs is unclear. In this study, we identified Y-box binding protein 1 (YBX1) as a carrier of SATII RNA via mass spectrometry analysis after RNA pull-down. sEVs containing SATII RNA induced cellular senescence and promoted the expression of inflammatory SASP genes in recipient cells. YBX1 knockdown significantly reduced SATII RNA levels in sEVs and inhibited the propagation of SASP in recipient cells. The analysis of the clinical dataset revealed that YBX1 expression is higher in cancer stroma than in normal stroma of breast and ovarian cancer tissues. Furthermore, high YBX1 expression was correlated with poor prognosis in breast and ovarian cancers. This study demonstrated that SATII RNA loading into sEVs is regulated via YBX1 and that YBX1 is a promising target in novel cancer therapy.

LncRNA JPX-Enriched Chromatin Microenvironment Mediates Vascular Smooth Muscle Cell Senescence and Promotes Atherosclerosis.

Senescence is a series of degenerative changes in the structure and physiological function of an organism. Whether JPX (just proximal to XIST)-a newly identified age-related noncoding RNA by us-is associated with atherosclerosis is still unknown. Our study was to investigate the role of JPX and provide insights into potential therapies targeting atherosclerosis. We analyzed clinical data from multiple tissues including meniscus tissue, leukemia cells, and peripheral blood monocytes to identify age-related noncoding RNAs in senescent vascular smooth muscle cells (VSMCs). The molecular mechanism of JPX was investigated by capture hybridization analysis of RNA targets and chromatin immunoprecipitation. IGVTools and real-time quantitative polymerase chain reaction were used to evaluate the JPX expression during phenotype regulation in age-related disease models. The therapeutic potential of JPX was evaluated after establishing an atherosclerosis model in smooth muscle-specific Jpx knockout mice. JPX expression was upregulated in activated ras allele (H-rasV12)-induced senescent VSMCs and atherosclerotic arteries. JPX knockdown substantially reduced the elevation of senescence-associated secretory phenotype (SASP) genes in senescent VSMCs. Cytoplasmic DNA leaked from mitochondria via mitochondrial permeability transition pore formed by VDAC1 (voltage-dependent anion channel 1) oligomer activates the STING (stimulator of interferon gene) pathway. JPX could act as an enhancer for the SASP genes and functions as a scaffold molecule through interacting with phosphorylated p65/RelA and BRD4 (bromodomain-containing protein 4) in chromatin remodeling complex, promoting the transcription of SASP genes via epigenetic regulation. Smooth muscle knockout of Jpx in ApoeKO mice resulted in a decrease in plaque area, a reduction in SASP gene expression, and a decrease in senescence compared with controls. As an enhancer RNA, JPX can integrate p65 and BRD4 to form a chromatin remodeling complex, activating SASP gene transcription and promoting cellular senescence. These findings suggest that JPX is a potential therapeutic target for the treatment of age-related atherosclerosis.

TEAD4 antagonizes cellular senescence by remodeling chromatin accessibility at enhancer regions

Dramatic alterations in epigenetic landscapes are known to impact genome accessibility and transcription. Extensive evidence demonstrates that senescent cells undergo significant changes in chromatin structure; however, the mechanisms underlying the crosstalk between epigenetic parameters and gene expression profiles have not been fully elucidated. In the present study, we delineate the genome-wide redistribution of accessible chromatin regions that lead to broad transcriptome effects during senescence. We report that distinct senescence-activated accessibility regions (SAAs) are always distributed in H3K27ac-occupied enhancer regions, where they are responsible for elevated flanking senescence-associated secretory phenotype (SASP) expression and aberrant cellular signaling relevant to SASP secretion. Mechanistically, a single transcription factor, TEAD4, moves away from H3K27ac-labled SAAs to allow for prominent chromatin accessibility reconstruction during senescence. The enhanced SAAs signal driven by TEAD4 suppression subsequently induces a robust increase in the expression of adjacent SASP genes and the secretion of downstream factors, which contribute to the progression of senescence. Our findings illustrate a dynamic landscape of chromatin accessibility following senescence entry, and further reveal an insightful function for TEAD4 in regulating the broad chromatin state that modulates the overall transcriptional program of SASP genes.

P12.01.A TUMOR CELL SENESCENCE AND IMMUNE MICROENVIRONMENT IN GLIOBLASTOMAS

Abstract BACKGROUND Cellular senescence (CS) is a state of irreversible cell cycle arrest, and the expression of p16INK4a in cells is one of the reliable markers for CS. However, senescent cells are metabolically active with the senescence-associated secretory phenotype (SASP), which can influence the tissue microenvironment by paracrine signaling to the adjacent tumor, non-tumor, and immune cells. MATERIAL AND METHODS In the present study, we evaluated p16INK4a expression in glioblastoma and investigated its association with CS and SASPs. We analyzed the expression of p16INK4a in 73 glioblastomas by immunostaining. To examine the association of p16INK4a expression and CS, we performed senescence-associated β-galactosidase (SA-β-Gal) staining, a standard marker of CS, using glioblastoma cell lines and fresh frozen tumor tissues. For SASPs analysis, RNA sequencing with Gene Set Enrichment Analysis (GSEA) was performed. RESULTS Among 73 glioblastomas, twenty-eight cases (38.4%) revealed diffuse strong p16INK4a expression in tumor cells. The glioblastoma with diffuse p16INK4a expression (GMDP) patients were younger (52.4 vs. 59.2 years) and showed prolonged overall survival (median: 25.5 vs. 12.3 months) compared to those harboring negative expression. In vitro analysis, p16INK4a over-expressed glioblastoma cell line showed increased expression of SA-β-Gal which indicates CS. In addition, fresh frozen tissues from GMDP also revealed SA-β-Gal positivity. RNA sequencing analysis revealed that splicing or protein biosynthetic genes were enriched in GMDP, and GSEA showed significant enrichment of SASP genes (false discovery rate <0.05), especially chemokines associated with monocytes and macrophages. CONCLUSION Our data suggest that increased expression of p16INK4a in glioblastoma is associated with tumor cell senescence, and SASPs from senescent tumor cells could be one of the crucial modulators in the tumor immune microenvironment.

Senescence-associated secretory phenotype constructed detrimental and beneficial subtypes and prognostic index for prostate cancer patients undergoing radical prostatectomy

BackgroundCellular senescence is growing in popularity in cancer. A dual function is played by the senescence-associated secretory phenotype (SASP) that senescent cells produce in the development of pro-inflammatory niches, tissue regeneration or destruction, senescence propagation, and malignant transformation. In this study, we conducted thorough bioinformatic analysis and meta-analysis to discover detrimental and beneficial subtypes and prognostic index for prostate cancer (PCa) patients using the experimentally confirmed SASP genes.MethodsWe identified differentially expressed and prognosis-related SASP genes and used them to construct two molecular subtypes and risk score. Another two external cohorts were used to confirm the prognostic effect of the above subtypes and risk score and meta-analysis was further conducted. Additionally, functional analysis, tumor stemness and heterogeneity and tumor microenvironment were also evaluated. We completed analyses using software R 3.6.3 and its suitable packages. Meta-analysis was performed by software Stata 14.0.ResultsThrough multivariate Cox regression analysis and consensus clustering analysis, we used VGF, IGFBP3 and ANG to establish detrimental and beneficial subtypes in the TCGA cohort, which was validated through other two independent cohorts. Meta-analysis showed that detrimental SASP group had significantly higher risk of biochemical recurrence (BCR) than beneficial SASP group (HR: 2.48). Moreover, we also constructed and validated risk score based on these genes to better guide clinical practice. DNA repair, MYC target, oxidative phosphorylation, proteasome and ribosome were highly enriched in detrimental SASP group. Detrimental SASP group had significantly higher levels of B cells, CD8+ T cells, homologous recombination deficiency, loss of heterozygosity, microsatellite instability, purity, tumor mutation burden, mRNAsi, differentially methylated probes and epigenetically regulated RNA expression than beneficial SASP group. The top mutation genes between detrimental and beneficial SASP groups were SPOP, FOXA1, KMT2C, APC, BSN, DNAH17, MYH6, EPPK1, ZNF536 and ZC3H13 with statistical significance.ConclusionsFrom perspective of SASP, we found detrimental and beneficial tumor subtypes which were closely associated with BCR-free survival for PCa patients, which might be important for the furture research in the field of PCa.

EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance.

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.

Identification of Apolipoprotein D as a Dermal Fibroblast Marker of Human Aging for Development of Skin Rejuvenation Therapy.

The current understanding of skin aging is that senescent fibroblasts accumulate within the dermis and subcutaneous fat to cause abnormal tissue remodeling and extracellular matrix dysfunction, triggering a senescence-associated secretory phenotype (SASP). A novel therapeutic approach to prevent skin aging is to specifically eliminate senescent dermal fibroblasts; this requires the identification of specific protein markers for senescent cells. Apolipoprotein D (ApoD) is involved in lipid metabolism and antioxidant responses and is abundantly expressed in tissues affected by age-related diseases such as Alzheimer's disease and atherosclerosis. However, its behavior and role in skin aging remain unclear. In this study, we examined whether ApoD functions as a marker of aging using human dermal fibroblast aging models. In cellular senescence models induced through replicative aging and ionizing radiation exposure, ApoD expression was upregulated at the gene and protein levels and correlated with senescence-associated β-galactosidase activity and the decreased uptake of the proliferation marker bromodeoxyuridine, which was concomitant with the upregulation of SASP genes. Furthermore, ApoD-positive cells were found to be more abundant in the aging human dermis using fluorescence flow cytometry. These results suggest that ApoD is a potential clinical marker for identifying aging dermal fibroblasts.

Senolytic drugs, dasatinib and quercetin, attenuate adipose tissue inflammation, and ameliorate metabolic function in old age.

Aging results in an elevated burden of senescent cells, senescence-associated secretory phenotype (SASP), and tissue infiltration of immune cells contributing to chronic low-grade inflammation and a host of age-related diseases. Recent evidence suggests that the clearance of senescent cells alleviates chronic inflammation and its associated dysfunction and diseases. However, the effect of this intervention on metabolic function in old age remains poorly understood. Here, we demonstrate that dasatinib and quercetin (D&Q) have senolytic effects, reducing age-related increase in senescence-associated β-galactosidase, expression of p16 and p21 gene and P16 protein in perigonadal white adipose tissue (pgWAT; all p≤ 0.04). This treatment also suppressed age-related increase in the expression of a subset of pro-inflammatory SASP genes (mcp1, tnf-α, il-1α, il-1β, il-6, cxcl2, and cxcl10), crown-like structures, abundance of T cells and macrophages in pgWAT (all p≤ 0.04). In the liver and skeletal muscle, we did not find a robust effect of D&Q on senescence and inflammatory SASP markers. Although we did not observe an age-related difference in glucose tolerance, D&Q treatment improved fasting blood glucose (p= 0.001) and glucose tolerance (p= 0.007) in old mice that was concomitant with lower hepatic gluconeogenesis. Additionally, D&Q improved insulin-stimulated suppression of plasma NEFAs (p= 0.01), reduced fed and fasted plasma triglycerides (both p≤ 0.04), and improved systemic lipid tolerance (p= 0.006). Collectively, results from this study suggest that D&Q attenuates adipose tissue inflammation and improves systemic metabolic function in old age. These findings have implications for the development of therapeutic agents to combat metabolic dysfunction and diseases in old age.

Abstract C015: EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype and promotes immune surveillance in PDAC

Abstract T-cell based immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that induction of cellular senescence and its accompanying senescence-associated secretory phenotype (SASP) can be a powerful way to enhance T cell infiltration into tumors and reactivate a different type of innate Natural Killer (NK) cell anti-tumor immunity that can mediate tumor control. Here, we found that the pancreas tumor microenvironment (TME) suppresses NK cell surveillance following therapy-induced senescence (TIS) with MEK and CDK4/6 inhibitor treatment through EZH2-mediated repression of pro-inflammatory SASP genes. Genetic or pharmacological inhibition of EZH2 or its methyltransferase activity enhanced the production of pro-inflammatory SASP factors and led to NK and T cell infiltration and immune-mediated tumor control in transplanted and genetically engineered PDAC mouse models. Mechanistically, EZH2 suppression induced expression of SASP factors CCL2 and CXCL9/10 necessary for lymophocyte chemotaxis into the pancreas TME. EZH2 levels were also associated with reduced NK cell numbers, SASP expression, and overall survival in a PDAC patient cohort. Taken together these results demonstrate that EZH2 mediates repression of the pro-inflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK and T cell surveillance in PDAC to achieve immune-mediated tumor responses. Citation Format: Loretah Chibaya, Katherine C. Murphy, Yvette Lopez Diaz, Kelly D. DeMarco, Haibo Liu, Sneha Gopalan, Melissa Faulkner, Junhui Li, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Elisa de Stanchina, Karl Simin, Lihua J. Zhu, Thomas G. Fazzio, Scott W. Lowe, Marcus Ruscetti. EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype and promotes immune surveillance in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C015.

TMIC-14. DIFFUSE P16INK4A EXPRESSION IS ASSOCIATED WITH TUMOR CELL SENESCENCE AND SENESCENCE-ASSOCIATED SECRETARY PHENOTYPE RELATED TO IMMUNE MICROENVIRONMENT IN GLIOBLASTOMAS

Abstract Cellular senescence (CS) is a state of irreversible cell cycle arrest, and the expression of p16INK4a in cells is one of the reliable markers for CS. However, senescent cells are metabolically active with the senescence-associated secretory phenotype (SASP), which can influence the tissue microenvironment by paracrine signaling to the adjacent tumor, non-tumor, and immune cells. In the present study, we evaluated p16INK4a expression in glioblastoma and investigated its association with CS and SASPs. We analyzed the expression of p16INK4a in 73 glioblastomas by immunostaining. To examine the association of p16INK4a expression and CS, we performed senescence-associated β-galactosidase (SA-β-Gal) staining, a standard marker of CS, using glioblastoma cell lines and fresh frozen tumor tissues. For SASPs analysis, RNA sequencing with Gene Set Enrichment Analysis (GSEA) was performed. Among 73 glioblastomas, twenty-eight cases (38.4%) revealed diffuse strong p16INK4a expression in tumor cells. The glioblastoma with diffuse p16INK4a expression (GMDP) patients were younger (52.4 vs. 59.2 years) and showed prolonged overall survival (median: 25.5 vs. 12.3 months) compared to those harboring negative expression. In vitro analysis, p16INK4a over-expressed glioblastoma cell line showed increased expression of SA-β-Gal which indicates CS. In addition, fresh frozen tissues from GMDP also revealed SA-β-Gal positivity. RNA sequencing analysis revealed that splicing or protein biosynthetic genes were enriched in GMDP, and GSEA showed significant enrichment of SASP genes (false discovery rate < 0.05), especially chemokines associated with monocytes and macrophages. Our data suggest that increased expression of p16INK4a in glioblastoma is associated with tumor cell senescence, and SASPs from senescent tumor cells could be one of the crucial modulators in the tumor immune microenvironment.

Role of Carbon Monoxide in Oxidative Stress-Induced Senescence in Human Bronchial Epithelium.

Prolonged or excessive stimulation from inhaled toxins may cause oxidative stress and DNA damage that can lead to stress-induced senescence in epithelial cells, which can contribute to several airway diseases. Mounting evidence has shown carbon monoxide (CO) confers cytoprotective effects. We investigated the effects of CO on oxidative stress-induced senescence in human airway epithelium and elucidated the underlying molecular mechanisms. Here, CO pretreatment reduced H2O2-mediated increases in total reactive oxygen species (ROS) production and mitochondrial superoxide in a human bronchial epithelial cell line (BEAS-2B). H2O2 treatment triggered a premature senescence-like phenotype with enlarged and flattened cell morphology accompanied by increased SA-β-gal activity, cell cycle arrest in G0/G1, reduced cell viability, and increased transcription of senescence-associated secretory phenotype (SASP) genes. Additionally, exposure to H2O2 increased protein levels of cellular senescence markers (p53 and p21), reduced Sirtuin 3 (SIRT3) and manganese superoxide dismutase (MnSOD) levels, and increased p53 K382 acetylation. These H2O2-mediated effects were attenuated by pretreatment with a CO-containing solution. SIRT3 silencing induced mitochondrial superoxide production and triggered a senescence-like phenotype, whereas overexpression decreased mitochondrial superoxide production and alleviated the senescence-like phenotype. Air-liquid interface (ALI) culture of primary human bronchial cells, which becomes a fully differentiated pseudostratified mucociliary epithelium, was used as a model. We found that apical and basolateral exposure to H2O2 induced a vacuolated structure that impaired the integrity of ALI cultures, increased goblet cell numbers, decreased SCGB1A1+ club cell numbers, increased p21 protein levels, and increased SASP gene transcription, consistent with our observations in BEAS-2B cells. These effects were attenuated in the apical presence of a CO-containing solution. In summary, we revealed that CO has a pivotal role in epithelial senescence by regulating ROS production via the SIRT3/MnSOD/p53/p21 pathway. This may have important implications in the prevention and treatment of age-associated respiratory pathologies.

Cathepsin F is a potential marker for senescent human skin fibroblasts and keratinocytes associated with skin aging

Cellular senescence is characterized by cell cycle arrest and the senescence-associated secretory phenotype (SASP) and can be triggered by a variety of stimuli, including deoxyribonucleic acid (DNA) damage, oxidative stress, and telomere exhaustion. Cellular senescence is associated with skin aging, and identification of specific markers of senescent cells is essential for development of targeted therapies. Cathepsin F (CTSF) has been implicated in dermatitis and various cancers and participates in cell immortalization through its association with Bcl family proteins. It is a candidate therapeutic target to specifically label and eliminate human skin fibroblasts and keratinocytes immortalized by aging and achieve skin rejuvenation. In this study, we investigated whether CTSF is associated with senescence in human fibroblasts and keratinocytes. In senescence models, created using replicative aging, ionizing radiation exposure, and the anticancer drug doxorubicin, various senescence markers were observed, such as senescence-associated β-galactosidase (SA-β-gal) activity, increased SASP gene expression, and decreased uptake of the proliferation marker BrdU. Furthermore, CTSF expression was elevated at the gene and protein levels. In addition, CTSF-positive cells were abundant in aged human epidermis and in some parts of the dermis. In the population of senescent cells with arrested division, the number of CTSF-positive cells was significantly higher than that in the proliferating cell population. These results suggest that CTSF is a candidate for therapeutic modalities targeting aging fibroblasts and keratinocytes.