Oncosuppressive role of MicroRNA-205–3p in gastric cancer through inhibition of proliferation and induction of senescence: Oncosuppressive role of MicroRNA-205 in gastric cancer

BackgroundOur previous study showed that CXCL11 could play an immunomodulatory role. In this study, we investigated the regulator (miR-205–3p) of CXCL11 and the mechanism of miR-205–3p as a tumor suppressor gene in gastric cancer (GC).Materials and methodsA target relationship between miR-205–3p and CXCL11 was revealed by using the bioinformatics method. This study detected the expressions of miR-205–3p and CXCL11 through qRT-PCR and Western blotting. Moreover, the expressions of Akt, PD-L1, p16, p21, and senescence-associated secretory phenotype (SASP) factor were determined. The effects of miR-205 on proliferation, invasion, and senescence of GC cells were assessed by using methods, such as transfection, Transwell assay, tablet cloning, flow cytometry, and senescence-associated beta-galactosidase (SA-β-gal) staining. Furthermore, the effects were verified using methods, like immunohistochemistry, flow cytometry and SA-β-gal in animal experiments.ResultsBased on the study, it is found that the expression of miR-205–3p is down-regulated, while that of CXCL11 is up-regulated in GC cell lines. By regulating CXCL11, miR-205–3p inhibits Akt activation, reduces the proliferation and invasion of GC cells, promotes cell apoptosis, induces senescence of GC cells, and secretes immunostimulatory SASP factor. The animal experiments confirm that miR-205–3p promotes cell senescence, down-regulates the immunosuppressive signal induced by PD-L1, and promotes secretion of immunostimulatory SASP factor, so that more T cells are recruited in blood and tumors.ConclusionsThis study revealed the molecular mechanism of miR-205–3p in inhibiting proliferation and invasion and inducing senescence of GC cells by regulating CXCL11 and Akt pathways in animal and cell experiments.Key words: microRNA-205–3p, CXCL11, Gastric cancer, Senescence, Proliferation     

Telomere Fragment Induced Amnion Cell Senescence: A Contributor to Parturition?

Oxidative stress (OS)-induced senescence of the amniochorion has been associated with parturition at term. We investigated whether telomere fragments shed into the amniotic fluid (AF) correlated with labor status and tested if exogenous telomere fragments (T-oligos) could induce human and murine amnion cell senescence. In a cross-sectional clinical study, AF telomere fragment concentrations quantitated by a validated real-time PCR assay were higher in women in labor at term compared to those not in labor. In vitro treatment of primary human amnion epithelial cells with 40 μM T-oligos ([TTAGGG]₂) that mimic telomere fragments, activated p38MAPK, produced senescence-associated (SA) β-gal staining and increased interleukin (IL)-6 and IL-8 production compared to cells treated with complementary DNA sequences (Cont-oligos, [AATCCC]₂). T-oligos injected into the uteri of pregnant CD1 mice on day 14 of gestation, led to increased p38MAPK, SA-β-gal (SA β-gal) staining in murine amniotic sacs and higher AF IL-8 levels on day 18, compared to saline treated controls. In summary, term labor AF samples had higher telomere fragments than term not in labor AF. In vitro and in situ telomere fragments increased human and murine amnion p38MAPK, senescence and inflammatory cytokines. We propose that telomere fragments released from senescent fetal cells are indicative of fetal cell aging. Based on our data, these telomere fragments cause oxidative stress associated damages to the term amniotic sac and force them to release other DAMPS, which, in turn, provide a sterile immune response that may be one of the many inflammatory signals required to initiate parturition at term.     

High Glucose Induced Alteration of SIRTs in Endothelial Cells Causes Rapid Aging in a p300 and FOXO Regulated Pathway

In diabetes, some of the cellular changes are similar to aging. We hypothesized that hyperglycemia accelerates aging-like changes in the endothelial cells (ECs) and tissues leading to structural and functional damage. We investigated glucose-induced aging in 3 types of ECs using senescence associated β-gal (SA β-gal) staining and cell morphology. Alterations of sirtuins (SIRTs) and their downstream mediator FOXO and oxidative stress were investigated. Relationship of such alteration with histone acetylase (HAT) p300 was examined. Similar examinations were performed in tissues of diabetic animals. ECs in high glucose (HG) showed evidence of early senescence as demonstrated by increased SA β-gal positivity and reduced replicative capacities. These alterations were pronounced in microvascular ECs. They developed an irregular and hypertrophic phenotype. Such changes were associated with decreased SIRT (1–7) mRNA expressions. We also found that p300 and SIRT1 regulate each other in such process, as silencing one led to increase of the others’ expression. Furthermore, HG caused reduction in FOXO1’s DNA binding ability and antioxidant target gene expressions. Chemically induced increased SIRT1 activity and p300 knockdown corrected these abnormalities slowing aging-like changes. Diabetic animals showed increased cellular senescence in renal glomerulus and retinal blood vessels along with reduced SIRT1 mRNA expressions in these tissues. Data from this study demonstrated that hyperglycemia accelerates aging-like process in the vascular ECs and such process is mediated via downregulation of SIRT1, causing reduction of mitochondrial antioxidant enzyme in a p300 and FOXO1 mediated pathway.     

Senescence induced by RECQL4 dysfunction contributes to Rothmund–Thomson syndrome features in mice

Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund–Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated β-galactosidase (SA-β-gal), higher expression of p16^INK4a or/and p21^WAF1 and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, Recql4-deficient mice (Recql4^HD). Tail fibroblasts from Recql4^HD showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in Recql4^HD mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients.     

Amyloid-β1–42 oligomer accelerates senescence in adult hippocampal neural stem/progenitor cells via formylpeptide receptor 2

The failure of adult hippocampal neurogenesis is increasingly considered as an important factor in the pathological correlates for memory decline in Alzheimer''s disease (AD). Loss of adult-born neurons and abnormalities of neural stem/progenitor cells (NSPCs) within the dentate gyrus (DG) of adult hippocampus might contribute to this process. In this study, we showed that amyloid-β_1–42 (Aβ₄₂) oligomer triggers senescent phenotype of NSPCs in vitro. Oligomerized Aβ₄₂ induced the production of senescence-associated biomarkers p16 and senescence-associated β-galactosidase (SA-β-gal) in adult mouse hippocampal NSPCs, as well as inhibited cells proliferation and differentiation. In the DG of amyloid precursor protein/presenilin1 (APP/PS1) transgenic mice, the number of senescent NSPCs was significantly increased and senescence-associated protein p16 was upregulated. Formylpeptide receptor 2 (FPR2), one of Aβ₄₂ functional receptors, may be involved in NSPCs senescence. The FPR2 antagonist WRW4 significantly inhibited NSPCs senescence induced by Aβ₄₂. In addition, the activation of p38 mitogen-activated protein kinase (MAPK) in response to the accumulation of reactive oxygen species (ROS) was involved in NSPCs senescence induced by Aβ₄₂. WRW4 inhibited the accumulation of ROS and the activation of p38 MAPK in NSPCs. Our data suggest that Aβ₄₂ accelerates NSPCs senescence via FPR2-dependent activation of its downstream ROS-p38 MAPK signaling, which limits the function of NSPCs and contributes to failure of neurogenesis. This is the first demonstration of NSPCs senescence response to Aβ₄₂.     

Upregulation of miR-760 and miR-186 Is Associated with Replicative Senescence in Human Lung Fibroblast Cells

We have previously shown that microRNAs (miRNAs) miR-760, miR-186, miR-337-3p, and miR-216b stimulate premature senescence through protein kinase CK2 (CK2) down-regulation in human colon cancer cells. Here, we examined whether these four miRNAs are involved in the replicative senescence of human lung fibroblast IMR-90 cells. miR-760 and miR-186 were significantly upregulated in replicatively senescent IMR-90 cells, and their joint action with both miR-337-3p and miR-216b was necessary for efficient downregulation of the α subunit of CK2 (CK2α) in IMR-90 cells. A mutation in any of the four miRNA-binding sequences within the CK2α 3′-untranslated region (UTR) indicated that all four miRNAs should simultaneously bind to the target sites for CK2α downregulation. The four miRNAs increased senescence-associated β-galactosidase (SA-β-gal) staining, p53 and p21^Cip1/WAF1 expression, and reactive oxygen species (ROS) production in proliferating IMR-90 cells. CK2α over-expression almost abolished this event. Taken together, the present results suggest that the upregulation of miR-760 and miR-186 is associated with replicative senescence in human lung fibroblast cells, and their cooperative action with miR-337-3p and miR-216b may induce replicative senescence through CK2α downregulation-dependent ROS generation.     

Effects of Cigarette Smoke Extracts on the Growth and Senescence of Skin Fibroblasts In Vitro

Epidemiological studies have shown that cigarette smoke (CS), a very common environmental factor, plays an important role in skin aging. Although some in vivo studies have suggested that CS affects skin aging, the detailed effects of CS on skin cells in vitro remain largely unknown. In this study, we investigated the effects of cigarette smoke extract (CSE) on the growth, proliferation, and senescene of skin fibroblasts and the possible mechanism underlying these effects. Primary cultured human fibroblasts were exposed to a range of concentrations of CSE. Cell viability and cell proliferation after CSE exposure were analyzed with the methyl thiazolyl tetrazolium (MTT) assay and bromodeoxyuridine incorporation assay, respectively. Growth curves of fibroblasts exposed to different concentrations of CSE were developed and prolonged CSE-exposed cells were observed. Morphological and ultrastructural changes in fibroblasts were assessed by inverted light microscopy and transmission electron microscopy (TEM). Dying cells were stained with senescence-associated β-galactosidase (SA β-gal). Intracellular reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity were determined by a colorimetric method. We found that proliferative capacity and growth were inhibited by CSE exposure in a dose- and time-dependent manner. Fibroblasts exposed to even low concentrations of CSE for a long period of time (5 passages) showed significantly increased SA β-gal activity and typical features of aging cells. Meanwhile, CSE inhibited superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and augmented ROS levels. Our observations suggest that CSE exposure impairs fibroblast growth and proliferation and leads to features similar to those seen in senescent cells. Oxidative stress injury and inhibition of antioxidant defense activity may be involved in CSE-induced fibroblast senescence.     

CDK5RAP2 loss-of-function causes premature cell senescence via the GSK3β/β-catenin-WIP1 pathway

Developmental disorders characterized by small body size have been linked to CDK5RAP2 loss-of-function mutations, but the mechanisms underlying which remain obscure. Here, we demonstrate that knocking down CDK5RAP2 in human fibroblasts triggers premature cell senescence that is recapitulated in Cdk5rap2^an/an mouse embryonic fibroblasts and embryos, which exhibit reduced body weight and size, and increased senescence-associated (SA)-β-gal staining compared to Cdk5rap2^+/+ and Cdk5rap2^+/an embryos. Interestingly, CDK5RAP2-knockdown human fibroblasts show increased p53 Ser15 phosphorylation that does not correlate with activation of p53 kinases, but rather correlates with decreased level of the p53 phosphatase, WIP1. Ectopic WIP1 expression reverses the senescent phenotype in CDK5RAP2-knockdown cells, indicating that senescence in these cells is linked to WIP1 downregulation. CDK5RAP2 interacts with GSK3β, causing increased inhibitory GSK3β Ser9 phosphorylation and inhibiting the activity of GSK3β, which phosphorylates β-catenin, tagging β-catenin for degradation. Thus, loss of CDK5RAP2 decreases GSK3β Ser9 phosphorylation and increases GSK3β activity, reducing nuclear β-catenin, which affects the expression of NF-κB target genes such as WIP1. Consequently, loss of CDK5RAP2 or β-catenin causes WIP1 downregulation. Inhibition of GSK3β activity restores β-catenin and WIP1 levels in CDK5RAP2-knockdown cells, reducing p53 Ser15 phosphorylation and preventing senescence in these cells. Conversely, inhibition of WIP1 activity increases p53 Ser15 phosphorylation and senescence in CDK5RAP2-depleted cells lacking GSK3β activity. These findings indicate that loss of CDK5RAP2 promotes premature cell senescence through GSK3β/β-catenin downregulation of WIP1. Premature cell senescence may contribute to reduced body size associated with CDK5RAP2 loss-of-function.Subject terms: Senescence, Diseases     

Overexpression of the Novel Senescence Marker β-Galactosidase (GLB1) in Prostate Cancer Predicts Reduced PSA Recurrence

Purpose

Senescence is a terminal growth arrest that functions as a tumor suppressor in aging and precancerous cells and is a response to selected anticancer compounds. Lysosomal-β-galactosidase (GLB1) hydrolyzes β-galactose from glycoconjugates and is the origin of senescence-associated β-gal activity (SA-β-gal). Using a new GLB1 antibody, senescence biology was investigated in prostate cancer (PCa) tissues.

Experimental Design

In vitro characterization of GLB1 was determined in primary prostate epithelial cell cultures passaged to replicative senescence and in therapy-induced senescence in PCa lines using chemotherapeutic agents. FFPE tissue microarrays were subjected to immunofluorescent staining for GLB1, Ki67 and HP1γ and automated quantitative imaging initially using AQUA in exploratory samples and Vectra in a validation series.

Results

GLB1 expression accumulates in replicative and induced senescence and correlates with senescent morphology and P16 (CDKN2) expression. In tissue arrays, quantitative imaging detects increased GLB1 expression in high-grade prostatic intraepithelial neoplasia (HGPIN), known to contain senescent cells, and cancer compared to benign prostate tissues (p<0.01) and senescent cells contain low Ki67 and elevated HP1γ. Within primary tumors, elevated GLB1 associates with lower T stage (p=0.01), localized versus metastatic disease (p=0.0003) and improved PSA-free survival (p=0.03). Increased GLB1 stratifies better PSA-free survival in intermediate grade PCa (0.01). Tissues that elaborate higher GLB1 display increased uniformity of expression.

Conclusion

Increased GLB1 is a valuable marker in formalin-fixed paraffin-embedded (FFPE) tissues for the senescence-like phenotype and associates with improved cancer outcomes. This protein addresses a lack of senescence markers and should be applicable to study the biologic role of senescence in other cancers.     

Shp2 signaling suppresses senescence in PyMT-induced mammary gland cancer in mice

In this study, we have used techniques from cell biology, biochemistry, and genetics to investigate the role of the tyrosine phosphatase Shp2 in tumor cells of MMTV-PyMT mouse mammary glands. Genetic ablation or pharmacological inhibition of Shp2 induces senescence, as determined by the activation of senescence-associated β-gal (SA-β-gal), cyclin-dependent kinase inhibitor 1B (p27), p53, and histone 3 trimethylated lysine 9 (H3K9me3). Senescence induction leads to the inhibition of self-renewal of tumor cells and blockage of tumor formation and growth. A signaling cascade was identified that acts downstream of Shp2 to counter senescence: Src, focal adhesion kinase, and Map kinase inhibit senescence by activating the expression of S-phase kinase-associated protein 2 (Skp2), Aurora kinase A (Aurka), and the Notch ligand Delta-like 1 (Dll1), which block p27 and p53. Remarkably, the expression of Shp2 and of selected target genes predicts human breast cancer outcome. We conclude that therapies, which rely on senescence induction by inhibiting Shp2 or controlling its target gene products, may be useful in blocking breast cancer.     

Resveratrol Induces Premature Senescence in Lung Cancer Cells via ROS-Mediated DNA Damage

Resveratrol (RV) is a natural component of red wine and grapes that has been shown to be a potential chemopreventive and anticancer agent. However, the molecular mechanisms underlying RV''s anticancer and chemopreventive effects are incompletely understood. Here we show that RV treatment inhibits the clonogenic growth of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Interestingly, the tumor-suppressive effect of low dose RV was not associated with any significant changes in the expression of cleaved PARP and activated caspase-3, suggesting that low dose RV treatment may suppress tumor cell growth via an apoptosis-independent mechanism. Subsequent studies reveal that low dose RV treatment induces a significant increase in senescence-associated β–galactosidase (SA-β-gal) staining and elevated expression of p53 and p21 in NSCLC cells. Furthermore, we show that RV-induced suppression of lung cancer cell growth is associated with a decrease in the expression of EF1A. These results suggest that RV may exert its anticancer and chemopreventive effects through the induction of premature senescence. Mechanistically, RV-induced premature senescence correlates with increased DNA double strand breaks (DSBs) and reactive oxygen species (ROS) production in lung cancer cells. Inhibition of ROS production by N-acetylcysteine (NAC) attenuates RV-induced DNA DSBs and premature senescence. Furthermore, we show that RV treatment markedly induces NAPDH oxidase-5 (Nox5) expression in both A549 and H460 cells, suggesting that RV may increase ROS generation in lung cancer cells through upregulating Nox5 expression. Together, these findings demonstrate that low dose RV treatment inhibits lung cancer cell growth via a previously unappreciated mechanism, namely the induction of premature senescence through ROS-mediated DNA damage.     

Chrysanthemum coronarium L. Protects against Premature Senescence in Human Endothelial Cells

The senescence of vascular endothelial cells (EC) leads to vascular dysfunction. However, the molecular mechanisms of EC senescence and its associated pathophysiological changes have not yet been clearly studied. This study sought to inspect the Chrysanthemum coronarium L. (CC) extract’s mechanism in preventing premature senescence of EC. A senescent endothelial cell model was created in human umbilical vein endothelial cells (HUVECs) with 100 µmol/L H₂O₂ treatment for 24 h. The effect of CC on senescent HUVECs was elucidated by measuring the activity of β-galactosidase (SA-β-gal), which exhibits an aging-related phenotype. SA-β-gal activity increased to 13.2 ± 2.85% in H₂O₂-treated HUVECs, whereas this activity was attenuated in the CC group. Immunoblot analyses revealed that p21, p53, and PAI-1 levels increased in the senescent HUVECs; however, the levels decreased in the HUVECs treated with various concentrations of CC (10, 20, and 50 μg/mL). The CC extract reduced the production of reactive oxygen species and reversed the decrease in NO production. Additionally, pretreatment with an Nω-nitro-l-arginine methyl ester (eNOS inhibitor) and nicotinamide (sirtuin 1 inhibitor) inhibited the anti-senescent effect of CC extract in HUVECs. Taken together, this study validated the novel endothelial protective effect of CC extract and its prevention of senescence in HUVECs through the mechanism regulated by eNOS and SIRT1 expression.     

Arsenite induces premature senescence via p53/p21 pathway as a result of DNA damage in human malignant glioblastoma cells

In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced γH2AX foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated β-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage. [BMB Reports 2014; 47(10): 575-580]     

Isolation of a Stable Subpopulation of Mobilized Dental Pulp Stem Cells (MDPSCs) with High Proliferation,Migration, and Regeneration Potential Is Independent of Age

Insights into the understanding of the influence of the age of MSCs on their cellular responses and regenerative potential are critical for stem cell therapy in the clinic. We have isolated dental pulp stem cells (DPSCs) subsets based on their migratory response to granulocyte-colony stimulating factor (G-CSF) (MDPSCs) from young and aged donors. The aged MDPSCs were efficiently enriched in stem cells, expressing high levels of trophic factors with high proliferation, migration and anti-apoptotic effects compared to young MDPSCs. In contrast, significant differences in those properties were detected between aged and young colony-derived DPSCs. Unlike DPSCs, MDPSCs showed a small age-dependent increase in senescence-associated β-galactosidase (SA-β-gal) production and senescence markers including p16, p21, Interleukin (IL)-1β, -6, -8, and Groα in long-term culture. There was no difference between aged and young MDPSCs in telomerase activity. The regenerative potential of aged MDPSCs was similar to that of young MDPSCs in an ischemic hindlimb model and an ectopic tooth root model. These results demonstrated that the stem cell properties and the high regenerative potential of MDPSCs are independent of age, demonstrating an immense utility for clinical applications by autologous cell transplantation in dental pulp regeneration and ischemic diseases.     

Ginsenoside Rb1 Prevents H2O2-Induced HUVEC Senescence by Stimulating Sirtuin-1 Pathway

Purposes

We have previously reported that Ginsenoside Rb1 may effectively prevent HUVECs from senescence, however, the detailed mechanism has not demonstrated up to now. Recent studies have shown that sirtuin-1 (Sirt1) plays an important role in the development of endothelial senescence. The purpose of this study was to explore whether Sirt1 is involved in the action of Ginsenoside Rb1 regarding protection against H₂O₂-induced HUVEC Senescence.

Methods and Results

Senescence induced by hydrogen peroxide (H₂O₂) in human umbilical vein endothelial cells (HUVECs) was examined by analyzing plasminogen activator inhibitor-1 (PAI-1) expression, cell morphology, and senescence-associated beta-galactosidase (SA-β-gal) activity. The results revealed that 42% of control-treated HUVECs were SA-β-gal positive after treatment by 60 µmol/L H₂O₂, however, this particular effect of H₂O₂ was decreased more than 2-fold (19%) in the HUVECs when pretreated with Rb1 (20 µmol/L) for 30 min. Additionally, Rb1 decreased eNOS acetylation, as well as promoted more NO production that was accompanied by an increase in Sirt1 expression. Furthermore, upon knocking down Sirt1, the effect of Rb1 on HUVEC senescence was blunted.

Conclusions

The present study indicated that Ginsenoside Rb1 acts through stimulating Sirt1 in order to protect against endothelial senescence and dysfunction. As such, Sirt1 appears to be of particular importance in maintaining endothelial functions and delaying vascular aging.     

Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides

Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence—as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21^{WAF1/Cip1/CDKN1A} (p21) expression, and senescence-associated β-galactosidase activity (SA-β-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-β-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.Subject terms: Cancer metabolism, Senescence