RESUMO
XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Síndrome de Cockayne/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Instabilidade Genômica , Recombinação Homóloga , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Síndrome de Cockayne/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi , Genoma Humano , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Fosforilação , Rad51 Recombinase/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
DNA damage can induce a tumor suppressive response termed cellular senescence. Damaged senescent cells permanently arrest growth, secrete inflammatory cytokines and other proteins and harbor persistent nuclear foci that contain DNA damage response (DDR) proteins. To understand how persistent damage foci differ from transient foci that mark repairable DNA lesions, we identify sequential events that differentiate transient foci from persistent foci, which we term 'DNA segments with chromatin alterations reinforcing senescence' (DNA-SCARS). Unlike transient foci, DNA-SCARS associate with PML nuclear bodies, lack the DNA repair proteins RPA and RAD51, lack single-stranded DNA and DNA synthesis and accumulate activated forms of the DDR mediators CHK2 and p53. DNA-SCARS form independently of p53, pRB and several other checkpoint and repair proteins but require p53 and pRb to trigger the senescence growth arrest. Importantly, depletion of the DNA-SCARS-stabilizing component histone H2AX did not deplete 53BP1 from DNA-SCARS but diminished the presence of MDC1 and activated CHK2. Furthermore, depletion of H2AX reduced both the p53-dependent senescence growth arrest and p53-independent cytokine secretion. DNA-SCARS were also observed following severe damage to multiple human cell types and mouse tissues, suggesting that they can be used in combination with other markers to identify senescent cells. Thus, DNA-SCARS are dynamically formed distinct structures that functionally regulate multiple aspects of the senescent phenotype.
Assuntos
Ciclo Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Cromatina/metabolismo , Citocinas/metabolismo , Dano ao DNA/efeitos da radiação , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Citocinas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Raios XRESUMO
Human embryonic stem cells (hESCs) hold promise for the treatment of many human pathologies. For example, hESCs and the neuronal stem cells (NSCs) and neurons derived from them have significant potential as transplantation therapies for a variety of neurodegenerative diseases. Two concerns about the use of hESCs and their differentiated derivatives are their ability to function and their ability to resist neoplastic transformation in response to stresses that inevitably arise during their preparation for transplantation. To begin to understand how these cells handle genotoxic stress, we examined the responses of hESCs and derived NSCs and neurons to ionizing radiation (IR). Undifferentiated hESCs were extremely sensitive to IR, with nearly all the cells undergoing cell death within 5-7 h. NSCs and neurons were substantially more resistant to IR, with neurons showing the most resistant. Of interest, NSCs that survived IR underwent cellular senescence and acquired astrocytic characteristics. Unlike IR-treated astrocytes, however, the NSC-derived astrocytic cells that survived IR did not display the typical pro-inflammatory, pro-carcinogenic senescence-associated secretory phenotype. These findings suggest distinct genotoxic stress-responses of hESCs and derived NSC and neuronal populations, and suggest that damaged NSCs, while failing to function, may not cause local inflammation.
Assuntos
Diferenciação Celular/efeitos da radiação , Senescência Celular , Dano ao DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos da radiação , Tolerância a Radiação , Astrócitos/citologia , Astrócitos/metabolismo , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos da radiação , Neurônios/citologia , Neurônios/efeitos da radiaçãoRESUMO
Exposure of MCF-7 breast tumor cells or HCT-116 colon carcinoma cells to clinically relevant concentrations of doxorubicin (Adriamycin; Farmitalia Research Laboratories, Milan, Italy) or camptothecin results in both autophagy and senescence. To determine whether autophagy is required for chemotherapy-induced senescence, reactive oxygen generation induced by Adriamycin was suppressed by N-acetyl cysteine and glutathione, and the induction of ataxia telangiectasia mutated, p53, and p21 was modulated pharmacologically and/or genetically. In all cases, autophagy and senescence were collaterally suppressed. The close association between autophagy and senescence indicated by these experiments reflects their collateral regulation via common signaling pathways. The potential relationship between autophagy and senescence was further examined through pharmacologic inhibition of autophagy with chloroquine and 3-methyl-adenine and genetic ablation of the autophagy-related genes ATG5 and ATG7. However, inhibition of autophagy by pharmacological and genetic approaches could not entirely abrogate the senescence response, which was only reduced and/or delayed. Taken together, our findings suggest that autophagy and senescence tend to occur in parallel, and furthermore that autophagy accelerates the development of the senescent phenotype. However, these responses are not inexorably linked or interdependent, as senescence can occur when autophagy is abrogated.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Camptotecina/farmacologia , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Doxorrubicina/farmacologia , Autofagia/genética , Western Blotting , Técnicas de Cultura de Células , Senescência Celular/genética , Citometria de Fluxo , Células HCT116 , Humanos , Células MCF-7 , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cellular senescence, which is associated with aging, is a process by which cells enter a state of permanent cell cycle arrest, therefore constituting a potent tumor suppressive mechanism. Recent studies show that, despite the beneficial effects of cellular senescence, senescent cells can also exert harmful effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescent-associated secretory phenotype (SASP), which entails a striking increase in the secretion of pro-inflammatory cytokines. Here, we summarize our knowledge of the SASP and the impact it has on tissue microenvironments and ability to stimulate tumor progression.
Assuntos
Senescência Celular/fisiologia , Citocinas/metabolismo , Inflamação/metabolismo , Neoplasias/metabolismo , Animais , Progressão da Doença , Humanos , Inflamação/imunologia , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
Large cell carcinoma (LCC) is a rare and aggressive lung cancer subtype with poor prognosis and no targeted therapies. Tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we identify MMP1 as overexpressed specifically in LCC cell lines, and we show that expression of MMP1 by LCC cells is necessary for induction of fibroblast senescence and consequent tumor promotion in both cell culture and mouse models. We also show that MMP1, in combination with TGF-ß1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-ß1-induced TAF senescence. Our results establish an entirely new role for MMP1 in cancer, and support a novel therapeutic strategy in LCC based on targeting senescent TAFs.
Assuntos
Fibroblastos Associados a Câncer/enzimologia , Carcinoma de Células Grandes/enzimologia , Proliferação de Células , Senescência Celular , Neoplasias Pulmonares/enzimologia , Metaloproteinase 1 da Matriz/metabolismo , Animais , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metaloproteinase 1 da Matriz/genética , Camundongos Nus , Estresse Oxidativo , Comunicação Parácrina , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Carga TumoralRESUMO
Neurodegeneration is a major age-related pathology. Cognitive decline is characteristic of patients with Alzheimer's and related dementias and cancer patients after chemo- or radio-therapies. A recently emerged driver of these and other age-related pathologies is cellular senescence, a cell fate that entails a permanent cell cycle arrest and pro-inflammatory senescence-associated secretory phenotype (SASP). Although there is a link between inflammation and neurodegenerative diseases, there are many open questions regarding how cellular senescence affects neurodegenerative pathologies. Among the various cell types in the brain, astrocytes are the most abundant. Astrocytes have proliferative capacity and are essential for neuron survival. Here, we investigated the phenotype of primary human astrocytes made senescent by X-irradiation, and identified genes encoding glutamate and potassium transporters as specifically downregulated upon senescence. This down regulation led to neuronal cell death in co-culture assays. Unbiased RNA sequencing of transcripts expressed by non-senescent and senescent astrocytes confirmed that glutamate homeostasis pathway declines upon senescence. Our results suggest a key role for cellular senescence, particularly in astrocytes, in excitotoxicity, which may lead to neurodegeneration including Alzheimer's disease and related dementias.
Assuntos
Doença de Alzheimer/genética , Astrócitos/metabolismo , Senescência Celular/genética , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sistema X-AG de Transporte de Aminoácidos/genética , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/patologia , Cultura Primária de Células , Raios XRESUMO
ERCC1 (excision repair cross complementing-group 1) is a mammalian endonuclease that incises the damaged strand of DNA during nucleotide excision repair and interstrand cross-link repair. Ercc1-/Δ mice, carrying one null and one hypomorphic Ercc1 allele, have been widely used to study aging due to accelerated aging phenotypes in numerous organs and their shortened lifespan. Ercc1-/Δ mice display combined features of human progeroid and cancer-prone syndromes. Although several studies report cellular senescence and apoptosis associated with the premature aging of Ercc1-/Δ mice, the link between these two processes and their physiological relevance in the phenotypes of Ercc1-/Δ mice are incompletely understood. Here, we show that ERCC1 depletion, both in cultured human fibroblasts and the skin of Ercc1-/Δ mice, initially induces cellular senescence and, importantly, increased expression of several SASP (senescence-associated secretory phenotype) factors. Cellular senescence induced by ERCC1 deficiency was dependent on activity of the p53 tumor-suppressor protein. In turn, TNFα secreted by senescent cells induced apoptosis, not only in neighboring ERCC1-deficient nonsenescent cells, but also cell autonomously in the senescent cells themselves. In addition, expression of the stem cell markers p63 and Lgr6 was significantly decreased in Ercc1-/Δ mouse skin, where the apoptotic cells are localized, compared to age-matched wild-type skin, possibly due to the apoptosis of stem cells. These data suggest that ERCC1-depleted cells become susceptible to apoptosis via TNFα secreted from neighboring senescent cells. We speculate that parts of the premature aging phenotypes and shortened health- or lifespan may be due to stem cell depletion through apoptosis promoted by senescent cells.
Assuntos
Apoptose/genética , Senescência Celular/genética , Proteínas de Ligação a DNA/deficiência , Endonucleases/deficiência , Fibroblastos/metabolismo , Pele/metabolismo , Células-Tronco/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Transdução de Sinais/genética , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cellular senescence entails an irreversible growth arrest that evolved in part to prevent cancer. Paradoxically, senescent cells secrete proinflammatory and growth-stimulatory molecules, termed the senescence-associated secretory phenotype (SASP), which is correlated with cancer cell proliferation in culture and xenograft models. However, at what tumor stage and how senescence and the SASP act on endogenous tumor growth in vivo is unknown. To understand the role of senescence in cancer etiology, we subjected p16-3MR transgenic mice, which permit the identification and selective elimination of senescent cells in vivo, to the well-established two-step protocol of squamous cell skin carcinoma, in which tumorigenesis is initiated by a carcinogen 7,12-dimethylbenz[α]anthracene, and then promoted by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that TPA promotes skin carcinogenesis by inducing senescence and a SASP. Systemic induction of senescence in nontumor-bearing p16-3MR mice using a chemotherapy followed by the two-step carcinogenesis protocol potentiated the conversion of benign papillomas to carcinomas by elevating p38MAPK and MAPK/ERK signaling. Ablation of senescent cells reduced p38MAPK and MAPK/ERK signaling, thereby preventing the progression of benign papillomas to carcinomas. Thus, we show for the first time that senescent cells are tumor promoters, not tumor initiators, and that they stimulate skin carcinogenesis by elevating p38MAPK and MAPK/ERK signaling. These findings pave the way for developing novel therapeutics against senescence-fueled cancers. SIGNIFICANCE: These findings identify chemotherapy-induced senescence as a culprit behind tumor promotion, suggesting that elimination of senescent cells after chemotherapy may reduce occurrence of second cancers decades later. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3606/F1.large.jpg.
Assuntos
Carcinogênese/metabolismo , Carcinoma de Células Escamosas/patologia , Senescência Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias Cutâneas/patologia , Animais , Carcinogênese/patologia , Carcinoma de Células Escamosas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/metabolismoRESUMO
Bloom syndrome (BS) is a hereditary disorder characterized by pre- and postnatal growth retardation, genomic instability, and cancer. BLM, the gene defective in BS, encodes a DNA helicase thought to participate in genomic maintenance. We show that BS human fibroblasts undergo extensive apoptosis after DNA damage specifically when DNA replication forks are stalled. Damage during S, but not G1, caused BLM to rapidly form foci with gammaH2AX at replication forks that develop DNA breaks. These BLM foci recruited BRCA1 and NBS1. Damaged BS cells formed BRCA1/NBS1 foci with markedly delayed kinetics. Helicase-defective BLM showed dominant-negative activity with respect to apoptosis, but not BRCA1/NBS1 recruitment, suggesting catalytic and structural roles for BLM. Strikingly, inactivation of p53 prevented the death of damaged BS cells and delayed recruitment of BRCA1/NBS1. These findings suggest that BLM is an early responder to damaged replication forks. Moreover, p53 eliminates cells that rapidly assemble BRCA1/NBS1 without BLM, suggesting that BLM is essential for timely BRCA1/NBS1 function.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteína BRCA1/fisiologia , Síndrome de Bloom/fisiopatologia , Proteínas de Ciclo Celular/fisiologia , DNA Helicases/fisiologia , Reparo do DNA/fisiologia , Proteínas Nucleares/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Apoptose/fisiologia , Síndrome de Bloom/patologia , Divisão Celular/fisiologia , Linhagem Celular Transformada , Células Cultivadas , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Fase G1/fisiologia , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fenótipo , RecQ Helicases , Fase S/fisiologia , Telomerase/genéticaRESUMO
Senescent cells secrete diverse array of proteins. One group of proteins, damage-associated molecular pattern (DAMP) proteins exhibit relocalization from inside to outside the cell. High Mobility Group Box 1 protein (HMGB1) is the founding DAMP member. HMGB1 relocalization from the nucleus provides a molecular signature during senescence. We provide distinct molecular techniques (immunofluorescence, immunohistochemistry, and Western blot assays) to assess HMGB1 relocalization during the initial stages of senescence.
Assuntos
Alarminas/metabolismo , Senescência Celular , Proteína HMGB1/metabolismo , Rim/metabolismo , Pele/metabolismo , Animais , Células Cultivadas , Rim/citologia , Camundongos , Camundongos Endogâmicos C57BL , Pele/citologiaRESUMO
Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent-associated secretory phenotype (SASP) and expression of p16INK4a in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16-3MR mice that allow elimination of p16INK4a -positive cells with exposure to ganciclovir, we show that impaired T-cell proliferation is partially reversed, mechanistically dependent on p16INK4a expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16INK4a expression. Our results provide molecular insight on how senescence-inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment.
Assuntos
Senescência Celular/efeitos da radiação , Radiação Ionizante , Baço/metabolismo , Baço/efeitos da radiação , Linfócitos T/imunologia , Linfócitos T/efeitos da radiação , Animais , Antivirais/uso terapêutico , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Proliferação de Células/efeitos da radiação , Células Cultivadas , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Ganciclovir/uso terapêutico , Vírus da Coriomeningite Linfocítica/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo , Rejuvenescimento/fisiologia , Baço/virologiaRESUMO
Processes that have been linked to aging and cancer include an inflammatory milieu driven by senescent cells. Senescent cells lose the ability to divide, essentially irreversibly, and secrete numerous proteases, cytokines and growth factors, termed the senescence-associated secretory phenotype (SASP). Senescent cells that lack p53 tumor suppressor function show an exaggerated SASP, suggesting the SASP is negatively controlled by p53. Here, we show that increased p53 activity caused by small molecule inhibitors of MDM2, which promotes p53 degradation, reduces inflammatory cytokine production by senescent cells. Upon treatment with the MDM2 inhibitors nutlin-3a or MI-63, human cells acquired a senescence-like growth arrest, but the arrest was reversible. Importantly, the inhibitors reduced expression of the signature SASP factors IL-6 and IL-1α by cells made senescent by genotoxic stimuli, and suppressed the ability of senescent fibroblasts to stimulate breast cancer cell aggressiveness. Our findings suggest that MDM2 inhibitors could reduce cancer progression in part by reducing the pro-inflammatory environment created by senescent cells.
Assuntos
Senescência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Compostos de Espiro/farmacologia , Proteína Supressora de Tumor p53/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Prepúcio do Pênis/citologia , Raios gama , Humanos , Interleucina-1alfa/antagonistas & inibidores , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmão/citologia , Masculino , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/agonistas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Loss of heterozygosity on human chromosome 3p21.3 is a frequent occurrence in many tumor types. In a previous study, our laboratory demonstrated that an 80-kb P1 clone from chromosome 3 suppresses the tumorigenicity of the mouse fibrosarcoma cell line A9. Two cDNAs corresponding to genes encoded on this P1 clone, semaphorin 3F (SEMA3F) and N23, were tested for their effects on in vitro and in vivo growth characteristics after transfection into mouse A9 cells. Transfection of SEMA3F cDNA resulted in complete loss of tumorigenicity in nude mice, whereas transfection of N23 had no effect. Moreover, SEMA3F also functioned to block apoptosis of transfected A9 cells treated with Taxol or Adriamycin. The human ovarian adenocarcinoma cell line HEY showed a similar result as A9 cells, but the small cell lung cancer line GLC45 was unaffected by expression of SEMA3F.
Assuntos
Cromossomos Humanos Par 3/genética , Genes Supressores de Tumor , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose/genética , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , DNA Complementar/genética , Feminino , Fibrossarcoma/genética , Fibrossarcoma/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , TransfecçãoRESUMO
Senescence in cancer cells acts as a tumor suppressor, whereas in fibroblasts enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of cancer subtypes. However, the presence of senescent TAFs in lung cancer remains undefined. We examined senescence in TAFs from primary lung cancer and paired control fibroblasts from unaffected tissue in three major histologic subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Three independent senescence markers (senescence-associated beta-galactosidase, permanent growth arrest and spreading) were consistently observed in cultured LCC-TAFs only, revealing a selective premature senescence. Intriguingly, SCC-TAFs exhibited a poor growth response in the absence of senescence markers, indicating a dysfunctional phenotype rather than senescence. Co-culturing normal fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to render fibroblasts senescent through oxidative stress, indicating that senescence in LCC-TAFs is driven by heterotypic signaling. In addition, senescent fibroblasts provided selective growth and invasive advantages to LCC cells in culture compared to normal fibroblasts. Likewise, senescent fibroblasts enhanced tumor growth and lung dissemination of tumor cells when co-injected with LCC cells in nude mice beyond the effects induced by control fibroblasts. These results define the subtype-specific aberrant phenotypes of lung TAFs, thereby challenging the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless of their subtype. Importantly, because LCC often distinguishes itself in the clinic by its aggressive nature, we argue that senescent TAFs may contribute to the selective aggressive behavior of LCC tumors.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Carcinoma de Células Grandes/metabolismo , Senescência Celular , Neoplasias Pulmonares/metabolismo , Miofibroblastos/metabolismo , Comunicação Parácrina , Animais , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Grandes/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Forma Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Progressão da Doença , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Miofibroblastos/patologia , Invasividade Neoplásica , Estresse Oxidativo , Fenótipo , Transdução de Sinais , Fatores de Tempo , Microambiente Tumoral , beta-Galactosidase/metabolismoRESUMO
Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ-like helicase and is thought to participate in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non-homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild-type (wt) WRN. WRN possesses both 3' --> 5' exonuclease and 3' --> 5' helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN-/-) complemented with either wtWRN, exonuclease-defective WRN (E-), helicase-defective WRN (H-) or exonuclease/helicase-defective WRN (E-H-). The single E-and H- mutants each partially complemented the NHEJ abnormality of WRN-/- cells. Strikingly, the E-H- double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E- and H- single mutants increased HR to levels higher than those restored by either E-H- or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events.
Assuntos
Senescência Celular/genética , DNA Helicases/deficiência , Reparo do DNA/genética , Síndrome de Werner/enzimologia , Síndrome de Werner/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Linhagem Celular Transformada , DNA/genética , DNA/metabolismo , Dano ao DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Fibroblastos/metabolismo , Humanos , Mutação/genética , RecQ Helicases , Recombinação Genética/genética , Síndrome de Werner/fisiopatologia , Helicase da Síndrome de WernerRESUMO
The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.
Assuntos
Interleucina-1alfa/metabolismo , Neoplasias da Próstata/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1alfa/genética , Interleucina-6/metabolismo , Masculino , Camundongos SCID , Mitoxantrona/farmacologia , NF-kappa B/metabolismo , Fenótipo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Interferência de RNA , RNA Mensageiro/metabolismo , Sirolimo/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Fatores de Tempo , Transcrição Gênica , Transfecção , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cellular senescence irreversibly arrests proliferation in response to potentially oncogenic stress. Senescent cells also secrete inflammatory cytokines such as IL-6, which promote age-associated inflammation and pathology. HMGB1 (high mobility group box 1) modulates gene expression in the nucleus, but certain immune cells secrete HMGB1 as an extracellular Alarmin to signal tissue damage. We show that nuclear HMGB1 relocalized to the extracellular milieu in senescent human and mouse cells in culture and in vivo. In contrast to cytokine secretion, HMGB1 redistribution required the p53 tumor suppressor, but not its activator ATM. Moreover, altered HMGB1 expression induced a p53-dependent senescent growth arrest. Senescent fibroblasts secreted oxidized HMGB1, which stimulated cytokine secretion through TLR-4 signaling. HMGB1 depletion, HMGB1 blocking antibody, or TLR-4 inhibition attenuated senescence-associated IL-6 secretion, and exogenous HMGB1 stimulated NF-κB activity and restored IL-6 secretion to HMGB1-depleted cells. Our findings identify senescence as a novel biological setting in which HMGB1 functions and link HMGB1 redistribution to p53 activity and senescence-associated inflammation.
Assuntos
Regulação da Expressão Gênica , Proteína HMGB1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Humanos , Inflamação , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar subnuclear redistribution in S phase and colocalize in nuclear foci. The co-localization was observed in mid- to late S phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain both protein markers of stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each, and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity, and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S phase that is, at least in part, performed coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.