Chemokine signaling via the CXCR2 receptor reinforces senescence.

Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.

Interplay between Homeobox proteins and Polycomb repressive complexes in p16INK4a regulation.

The INK4/ARF locus regulates senescence and is frequently altered in cancer. In normal cells, the INK4/ARF locus is found silenced by Polycomb repressive complexes (PRCs). Which are the mechanisms responsible for the recruitment of PRCs to INK4/ARF and their other target genes remains unclear. In a genetic screen for transcription factors regulating senescence, we identified the homeodomain-containing protein HLX1 (H2.0-like homeobox 1). Expression of HLX1 extends cellular lifespan and blunts oncogene-induced senescence. Using quantitative proteomics, we identified p16(INK4a) as the key target mediating the effects of HLX1 in senescence. HLX1 represses p16(INK4a) transcription by recruiting PRCs and HDAC1. This mechanism has broader implications, as HLX1 also regulates a subset of PRC targets besides p16(INK4a). Finally, sampling members of the Homeobox family, we identified multiple genes with ability to repress p16(INK4a). Among them, we found HOXA9 (Homeobox A9), a putative oncogene in leukaemia, which also recruits PRCs and HDAC1 to regulate p16(INK4a). Our results reveal an unexpected and conserved interplay between homeodomain-containing proteins and PRCs with implications in senescence, development and cancer.

Coagulation and platelet biology at the intersection of health and disease: illustrated capsules of the 11th Symposium on Hemostasis at the University of North Carolina.

The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field. This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks.

The potential of aging rejuvenation.

Aging is a process by which basic cellular functions and tissue homeostasis start to decline and organs become progressively dysfunctional. Although aging was once considered irreversible, the concept of the elixir of youth or rejuvenation has been in the history for centuries. In fact, recent scientific studies now show the existence of alternative strategies to delay aging. Here, we discuss how different signaling pathways, a variety of cell types and molecules can contribute to delay aging. In addition, we will define recently described rejuvenation strategies, with an emphasis on the potential for extracellular vesicles (EV).

Cellular Senescence and Ageing: Mechanisms and Interventions.

The influence of the activation of a cellular phenotype termed senescence and it's importance in ageing and age-related diseases is becoming more and more evident. In fact, there is a huge effort to tackle these diseases via therapeutic drugs targeting senescent cells named senolytics. However, a clearer understanding of how senescence is activated and the influence it has on specific cellular types and tissues is needed. Here, we describe general triggers and characteristics of senescence. In addition, we describe the influence of senescent cells in ageing and different age-related diseases.

Genome wide CRISPR/Cas9 screen identifies the coagulation factor IX (F9) as a regulator of senescence.

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.

NF-κB/IKK activation by small extracellular vesicles within the SASP.

Cellular senescence plays an important role in different biological and pathological conditions. Senescent cells communicate with their microenvironment through a plethora of soluble factors, metalloproteases and extracellular vesicles (EV). Although much is known about the role that soluble factors play in senescence, the downstream signalling pathways activated by EV in senescence is unknown. To address this, we performed a small molecule inhibitor screen and have identified the IκB kinases IKKε, IKKα and IKKß as essential for senescence mediated by EV (evSASP). By using pharmacological inhibitors of IKKε, IKKα and IKKß, in addition to CRISPR/Cas9 targeting their respective genes, we find these pathways are important in mediating senescence. In addition, we find that senescence activation is dependent on canonical NF-κB transcription factors where siRNA targeting p65 prevent senescence. Importantly, these IKK pathways are also relevant to ageing as knockout of IKKA, IKKB and IKKE avoid the activation of senescence. Altogether, these findings open a new potential line of investigation in the field of senescence by targeting the negative effects of the evSASP independent of particular EV contents.

Surface-Enhanced Raman Scattering (SERS) Spectroscopy for Sensing and Characterization of Exosomes in Cancer Diagnosis.

Exosomes are emerging as one of the most intriguing cancer biomarkers in modern oncology for early cancer diagnosis, prognosis and treatment monitoring. Concurrently, several nanoplasmonic methods have been applied and developed to tackle the challenging task of enabling the rapid, sensitive, affordable analysis of exosomes. In this review, we specifically focus our attention on the application of plasmonic devices exploiting surface-enhanced Raman spectroscopy (SERS) as the optosensing technique for the structural interrogation and characterization of the heterogeneous nature of exosomes. We summarized the current state-of-art of this field while illustrating the main strategic approaches and discuss their advantages and limitations.

Therapy-Induced Senescence: Opportunities to Improve Anticancer Therapy.

Cellular senescence is an essential tumor suppressive mechanism that prevents the propagation of oncogenically activated, genetically unstable, and/or damaged cells. Induction of tumor cell senescence is also one of the underlying mechanisms by which cancer therapies exert antitumor activity. However, an increasing body of evidence from preclinical studies demonstrates that radiation and chemotherapy cause accumulation of senescent cells (SnCs) both in tumor and normal tissue. SnCs in tumors can, paradoxically, promote tumor relapse, metastasis, and resistance to therapy, in part, through expression of the senescence-associated secretory phenotype. In addition, SnCs in normal tissue can contribute to certain radiation- and chemotherapy-induced side effects. Because of its multiple roles, cellular senescence could serve as an important target in the fight against cancer. This commentary provides a summary of the discussion at the National Cancer Institute Workshop on Radiation, Senescence, and Cancer (August 10-11, 2020, National Cancer Institute, Bethesda, MD) regarding the current status of senescence research, heterogeneity of therapy-induced senescence, current status of senotherapeutics and molecular biomarkers, a concept of "one-two punch" cancer therapy (consisting of therapeutics to induce tumor cell senescence followed by selective clearance of SnCs), and its integration with personalized adaptive tumor therapy. It also identifies key knowledge gaps and outlines future directions in this emerging field to improve treatment outcomes for cancer patients.

Classical and Nonclassical Intercellular Communication in Senescence and Ageing.

Intercellular communication refers to the different ways through which cells communicate with each other and transfer a variety of messages. These communication methods involve a number of different processes that occur individually or simultaneously, which change depending on the physiological or pathological context. The best characterized means of intercellular communication is the release of soluble factors that affect the function of neighboring cells. However, there are many other ways by which cells can communicate with each other. Here, we review the different means of intercellular communication including soluble factors in the context of senescence, ageing, and age-related diseases.

Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage.

Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging.

Extracellular vesicles as potential tools for regenerative therapy.

Small extracellular vesicles released by fibroblasts from young human donors diminish lipid peroxidation in senescent cells and in different old mice organs due to their enrichment in Glutathione-S-transferase Mu lipid antioxidant activity.

Prominent role of NF-kappaB in the induction of endothelial activation by endogenous nitric oxide inhibition.

Decreased endothelial nitric oxide (NO) production and increased expression of vascular cell adhesion molecule-1 (VCAM-1) are early features of atherosclerosis. We investigated the effects of suppressing endogenous NO production by the NO synthase inhibitor l-mono-methyl-arginine (L-NMMA), given alone or in combination with interleukin(IL)-1alpha, on VCAM-1 expression by human umbilical vein endothelial cells (HUVEC). VCAM-1 expression (by enzyme immunoassay), barely detectable at baseline, was significantly increased by L-NMMA (by no more than 20% over control compared with IL-1alpha induction). This was paralleled by an increase in U937 monocytoid cell adhesion. When HUVEC incubated with L-NMMA were stimulated with low concentrations of IL-1alpha (0.05-0.5ng/mL), these determined a higher VCAM-1 expression than in the presence of L-NMMA or IL-1alpha alone. Northern analysis indicated that VCAM-1 mRNA was induced by L-NMMA alone, and that the effects of L-NMMA and IL-1alpha were, again, at least additive. Nuclear factor-kappaB (NF-kappaB), GATA, activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF-1), transcription factors all involved in VCAM-1 gene expression, were all activated at electrophoretic mobility shift assay and at chromatin immunoprecipitation assay by L-NMMA, but additive effects with the combined administration of L-NMMA and IL-1alpha only occurred for NF-kappaB. These results support the view that endogenous NO mantains a normal endothelial non-reactivity towards circulating monocytes, and that suppression of this endogenous brake for endothelial activation results in the activation of multiple transcription factors even in the absence of other endothelial activators, with a prominent role of NF-kappaB in the presence or absence of other inflammatory mediators.

Negative regulation of cell-cycle progression by RINGO/Speedy E.

Cell-cycle transitions are controlled by CDKs (cyclin-dependent kinases), whose activation is usually associated with the binding of cyclins. RINGO/Speedy proteins can also bind to and activate CDKs, although they do not have amino acid sequence homology with cyclins. The RINGO/Speedy family members studied so far positively regulate cell-cycle progression. In the present paper, we report the biochemical and functional characterization of RINGO/Speedy E. We show that RINGO/Speedy E is a functionally distant member of this protein family that negatively affects cell-cycle progression. RINGO/Speedy E overexpression inhibits the meiotic progression in Xenopus oocytes as well as the proliferation of mammalian cells. RINGO/Speedy E can bind to endogenous CDK1 and CDK2 in both cellular systems. However, the RINGO/Speedy E-activated CDKs have different substrate specificity than the CDKs activated by other RINGO/Speedy proteins, which may account for their different effects on the cell cycle. Our results indicate that, although all RINGO/Speedy family members can activate CDKs, they may differently regulate cell-cycle progression.

FASN activity is important for the initial stages of the induction of senescence.

Senescent cells accumulate in several tissues during ageing and contribute to several pathological processes such as ageing and cancer. Senescence induction is a complex process not well defined yet and is characterized by a series of molecular changes acquired after an initial growth arrest. We found that fatty acid synthase (FASN) levels increase during the induction of senescence in mouse hepatic stellate cells and human primary fibroblasts. Importantly, we also observed a significant increase in FASN levels during ageing in mouse liver tissues. To probe the central role of FASN in senescence induction, we used a small-molecule inhibitor of FASN activity, C75. We found that C75 treatment prevented the induction of senescence in mouse and human senescent cells. Importantly, C75 also reduced the expression of the signature SASP factors interleukin 1α (IL-1α), IL-1ß and IL-6, and suppressed the secretion of small extracellular vesicles. These findings were confirmed using a shRNA targeting FASN. In addition, we find that FASN inhibition induces metabolic changes in senescent cells. Our work underscores the importance of C75 as a pharmacological inhibitor for reducing the impact of senescent cell accumulation.

Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3.

Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.

Characterization of the activity of human MAP kinase-interacting kinase Mnk1b.

Human mitogen-activated protein (MAP) kinase interacting kinase 1b (Mnk1b) is a splice variant of human Mnk1a, which has been identified in our laboratory [A. O'Loghlen, V.M. Gonzalez, D. Pineiro, M.I. Perez-Morgado, M. Salinas, M.E. Martin, Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1, Exp. Cell Res. 299 (2004) 343-355]. Mnk1b has much higher basal eIF4E kinase activity than Mnk1a. Because Mnk1b presents different features in its C-terminus with respect to Mnk1a, we have studied in this paper the potential role of these structural differences in determining the higher basal eIF4E kinase activity as well as the subcellular localization of Mnk1b. In this paper, we demonstrate that phosphorylation of the Thr209 and Thr214 in the activation loop of Mnk1b is necessary for its activation. However, the different kinase activity between Mnk1a and Mnk1b is independent of the phosphorylation status of the activation loop residues. By deletion of the C-terminal tail in Mnk1a, we confirmed that the absence of this sequence is not responsible for the higher eIF4E kinase activity present in Mnk1b. Moreover, our findings support a crucial role of the 12 amino acids, particularly the Ala344, in the C-terminal specific region of Mnk1b (Mnk1bSR), on the kinase activity of the protein.

Role for extracellular vesicles in the tumour microenvironment.

Extracellular vesicles (EVs) are small-membrane vesicles secreted by most cells types with the role to provide intercellular communication both locally and systemically. The transfer of their content between cells, which includes nucleic acids, proteins and lipids, confers the means for these interactions and induces significant cellular behaviour changes in the receiving cell. EVs are implicated in the regulation of numerous physiological and pathological processes, including development and neurological and cardiovascular diseases. Importantly, it has been shown that EV signalling is essential in almost all the steps necessary for the progress of carcinomas, from primary tumours to metastasis. In this review, we will focus on the latest findings for EV biology in relation to cancer progression and the tumour microenvironment.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.

Technical Advances to Study Extracellular Vesicles.

Extracellular vesicles are a heterogeneous and dynamic group of lipid bilayer membrane nanoparticles that can be classified into three different groups depending on their cellular origin: exosomes, microvesicles, and apoptotic bodies. They are produced by different cell types and can be isolated from almost all body fluids. EVs contain a variety of proteins, lipids, nucleic acids, and metabolites which regulate a number of biological and pathological scenarios both locally and systemically. Different techniques have been described in order to determine EV isolation, release, uptake, and cargo. Although standard techniques such as immunoblotting, fluorescent microscopy, and electron microscopy are still being used to characterize and visualize EVs, in the last years, more fine-tuned techniques are emerging. For example, EV uptake can be specifically determined at a single cell level using the Cre reporter methodology and bioluminescence based-methods reports have been employed to determine both EV release and uptake. In addition, techniques for cargo identification have also enormously evolved during these years. Classical mass spectrometry and next generation sequencing have been used in the past, but nowadays, advances in these tools have facilitated a more in depth characterization of the EV content. In this review, we aim to assess the standard and latest technical advances for studying EV biology in different biological systems.

Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-ß Pathway.

Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or ß3) is regulated by the Polycomb protein CBX7. ß3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor ß (TGF-ß) pathway in a cell-autonomous and non-cell-autonomous manner. ß3 levels are dynamically increased during oncogene-induced senescence (OIS) through CBX7 Polycomb regulation, and downregulation of ß3 levels overrides OIS and therapy-induced senescence (TIS), independently of its ligand-binding activity. Moreover, cilengitide, an αvß3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP) without affecting proliferation. Finally, we show an increase in ß3 levels in a subset of tissues during aging. Altogether, our data show that integrin ß3 subunit is a marker and regulator of senescence.