RESUMEN
Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated ß-galactosidase (SA-ß-gal), the DNA synthesis marker 5-ethynyl-2'-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (γH2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.
Asunto(s)
Senescencia Celular , Neoplasias , Biomarcadores , Senescencia Celular/fisiología , Daño del ADN , Citometría de Flujo , Humanos , Neoplasias/patología , Microambiente Tumoral , beta-Galactosidasa/genéticaRESUMEN
Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19/patología , COVID-19/virología , Senescencia Celular/efectos de los fármacos , Terapia Molecular Dirigida , SARS-CoV-2/patogenicidad , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Animales , COVID-19/complicaciones , Línea Celular , Cricetinae , Dasatinib/farmacología , Dasatinib/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Quercetina/farmacología , Quercetina/uso terapéutico , SARS-CoV-2/efectos de los fármacos , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Trombosis/complicaciones , Trombosis/inmunología , Trombosis/metabolismoRESUMEN
Aberrant B-cell receptor/NF-κB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas, especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, for example, in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-κB, but their differential impact on lymphoma development and biology remains to be determined. Here, we functionally investigate primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells stably transduced with naturally occurring NF-κB mutants. Although most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced transforming growth factor ß (TGF-ß)-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune-checkpoint mediator programmed cell death ligand 1 (PD-L1), thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-programmed cell death 1 checkpoint blockade, leading to direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies.
Asunto(s)
Inmunidad Adaptativa , Proteínas Adaptadoras de Señalización CARD/genética , Senescencia Celular/fisiología , Guanilato Ciclasa/genética , Linfoma de Células B Grandes Difuso/inmunología , Factor 88 de Diferenciación Mieloide/genética , Proteínas de Neoplasias/genética , Linfocitos T Citotóxicos/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígenos CD79/genética , Línea Celular Tumoral , Quimiotaxis , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Genes Reporteros , Genes myc , Humanos , Inhibidores de Puntos de Control Inmunológico , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/terapia , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , FN-kappa B/genética , FN-kappa B/metabolismo , Mutación Puntual , Proteína 2 Ligando de Muerte Celular Programada 1/antagonistas & inhibidores , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , TranscriptomaRESUMEN
Lesion-based targeting strategies underlie cancer precision medicine. However, biological principles - such as cellular senescence - remain difficult to implement in molecularly informed treatment decisions. Functional analyses in syngeneic mouse models and cross-species validation in patient datasets might uncover clinically relevant genetics of biological response programs. Here, we show that chemotherapy-exposed primary Eµ-myc transgenic lymphomas - with and without defined genetic lesions - recapitulate molecular signatures of patients with diffuse large B-cell lymphoma (DLBCL). Importantly, we interrogate the murine lymphoma capacity to senesce and its epigenetic control via the histone H3 lysine 9 (H3K9)-methyltransferase Suv(ar)39h1 and H3K9me3-active demethylases by loss- and gain-of-function genetics, and an unbiased clinical trial-like approach. A mouse-derived senescence-indicating gene signature, termed "SUVARness", as well as high-level H3K9me3 lymphoma expression, predict favorable DLBCL patient outcome. Our data support the use of functional genetics in transgenic mouse models to incorporate basic biology knowledge into cancer precision medicine in the clinic.
Asunto(s)
Senescencia Celular , Histona Metiltransferasas , Linfoma de Células B Grandes Difuso , Células 3T3 , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Transgénicos , PronósticoRESUMEN
A cell's genomic integrity is at risk when DNA-damaging stress, evoked by mitogenic oncogenes or genotoxic treatment modalities such as radiation or chemotherapy, apply. If the DNA repair machinery fails to fix the damaged site during a temporary cell-cycle arrest, or if massive genotoxic stress overwhelmed the repair capacity, cellular failsafe programs such as apoptosis or senescence will be triggered to limit aberrant propagation of these damaged and potentially harmful cells. After decades of scientific focusing on apoptosis, cellular senescence is increasingly recognized as an equally important but biologically and fundamentally different type of ultimate cell-cycle exit program, because of its lastingly persistent nature and cell-intrinsic and extrinsic roles within the tissue and tumor microenvironment. We established primary apoptosis-compromised, Bcl2-expressing Eµ-myc transgenic mouse lymphomas as a versatile and clinically relevant model system to study therapy-induced senescence (TIS). Given the lack of a single specific senescence-defining marker, we previously exploited co-staining of senescence-associated ß-galactosidase (SA-ß-gal) activity with immunohistochemical detection of trimethylated histone H3 lysine 9 (H3K9me3), an established S-phase gene expression-controlling, repressive chromatin mark, and the proliferation marker Ki67. This biomarker panel is instrumental to characterize cells as senescent via their high SA-ß-gal activity, strong nuclear H3K9me3 expression and Ki67-negative profile. In this chapter, we demonstrate the detection of viable senescent cells by novel methods based on a fluorescent version of the SA-ß-gal (fSA-ß-gal) assay, combined with immuno-fluoroscence staining of H3K9me3 or Ki67, or analysis of the DNA replication status by incorporating 5-ethynyl-2'-deoxyuridine (EdU) detection into the protocol. Notably, while most senescence markers, irrespective of their specificity and sensitivity, may only be assessed in endpoint assays, we would like to emphasize here the strength of viable fSA-ß-gal to track single-cell fate in senescent populations over time.
Asunto(s)
Senescencia Celular , Daño del ADN , Fluorescencia , Linfoma/patología , beta-Galactosidasa/metabolismo , Animales , Apoptosis , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacología , Humanos , Linfoma/tratamiento farmacológico , Linfoma/enzimología , Linfoma/genética , Ratones , Ratones TransgénicosRESUMEN
Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eµ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.
Asunto(s)
Reprogramación Celular , Senescencia Celular , Linfoma de Células B/patología , Células Madre Neoplásicas/patología , Animales , Biomarcadores/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Reprogramación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Células Clonales/efectos de los fármacos , Células Clonales/patología , Femenino , Humanos , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/genética , Masculino , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/efectos de los fármacos , Fenotipo , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Therapy-induced senescence (TIS), a lasting chemotherapy-evoked proliferative arrest of tumor cells, has gained increasing attention by cancer researchers because of its' profound biological implications, and by clinical oncologists due to its potential contribution to the long-term outcome of cancer patients post-treatment. Although both apoptosis and senescence represent therapy-inducible, ultimate cell-cycle exit programs, mediated via DNA damage response signaling, apoptotic cell death as the faster and often quantitatively more prominent tumor response has been in the scientific focus for decades. The more recently recognized TIS as another "safeguard" response of cancer cells that were never primed for or failed to execute apoptosis, not only reflects a more complex "arrest-plus-other features" cell-autonomous condition but produces non-cell-autonomous phenotypes at the tumor site, collectively impinging on tumor control and clinical outcome. Hence, TIS research is gaining pivotal interest from both a tumor biological and a therapeutic perspective, and the development of non-DNA damaging, senescence-evoking therapeutics is about to become a major research objective. In this chapter, we describe a well-characterized, genetically controlled TIS model system based on primary BCL2-expressing Eµ-myc transgenic lymphoma cells harboring defined genetic lesions and provide protocols for co-staining of either senescence-associated ß-galactosidase (SA-ß-gal) activity or trimethylated lysine 9 of histone H3 (H3K9me3) together with Ki67 to detect the senescent status of therapy-exposed cancer cells.
Asunto(s)
Biomarcadores , Senescencia Celular , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Ensamble y Desensamble de Cromatina , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Sustitución del Gen , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Inmunohistoquímica , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common human cancers. Recently, emerging evidence has suggested the role of long non-coding RNAs (lncRNAs) in human carcinogenesis. In this study, we aimed to investigate the expression and functional implications of lncRNAs in human HCC. METHODS: Eighty-eight well-annotated lncRNAs were profiled in primary HCC by quantitative RT-PCR. Functional relevance of lncRNAs was elucidated in HCC cell lines and nude mice models. The regulatory relationship between miRNA and lncRNA was predicted in silico and further validated by luciferase reporter assay and expression analysis. RESULTS: In our profiling study, HOTTIP was identified as the most significantly up-regulated lncRNA in human HCCs, even in early stage of HCC formation. Functionally, knock-down of HOTTIP attenuated HCC cell proliferation in vitro and markedly abrogated tumourigenicity in vivo. In addition, knock-down of HOTTIP also inhibited migratory ability of HCC cells and significantly abrogated lung metastasis in orthotopic implantation model in nude mice. HOTTIP is an antisense lncRNA mapped to the distal end of the HOXA gene cluster. Knock-down of HOTTIP significantly suppressed the expression of a number of HOXA genes. Furthermore, we identified miR-125b as a post-transcriptional regulator of HOTTIP. Ectopic expression of miR-125b reduced HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Moreover, in human HCCs, HOTTIP expression negatively correlated with that of miR-125b. CONCLUSIONS: HOTTIP is a novel oncogenic lncRNA, which negatively regulated by miR-125b. Overexpression of HOTTIP contributes to hepatocarcinogenesis by regulating the expression of its neighbouring protein-coding genes.