RESUMO
Developmental senescence is a form of programmed senescence that contributes to morphogenesis during embryonic development. We showed recently that the SIX1 homeoprotein, an essential regulator of organogenesis, is also a repressor of adult cellular senescence. Alterations in the SIX/EYA pathway are linked to the human branchio-oto-renal (BOR) syndrome, a rare congenital disorder associated with defects in the ears, kidneys and branchial arches. Here, we have used Six1-deficient mice, an animal model of the BOR syndrome, to investigate whether dysfunction of senescence underpins the developmental defects associated with SIX1 deficiency. We have focused on the developing inner ear, an organ with physiological developmental senescence that is severely affected in Six1-deficient mice and BOR patients. We show aberrant levels and distribution of senescence markers in Six1-deficient inner ears concomitant with defective morphogenesis of senescent structures. Transcriptomic analysis and ex vivo assays support a link between aberrant senescence and altered morphogenesis in this model, associated with deregulation of the TGFß/BMP pathway. Our results show that misregulation of embryo senescence may lead to genetic developmental disorders, significantly expanding the connection between senescence and disease.
Assuntos
Síndrome Brânquio-Otorrenal , Orelha Interna , Adulto , Humanos , Camundongos , Animais , Proteínas Tirosina Fosfatases/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/genética , Síndrome Brânquio-Otorrenal/genética , Proteínas de Homeodomínio/metabolismoRESUMO
Cellular senescence is an antiproliferative response with a critical role in the control of cellular balance in diverse physiological and pathological settings. Here, we set to study the impact of senescence on the regulation of cell plasticity, focusing on the regulation of the myofibroblastic phenotype in primary fibroblasts. Myofibroblasts are contractile, highly fibrogenic cells with key roles in wound healing and fibrosis. Using cellular models of fibroblast senescence, we find a consistent loss of myofibroblastic markers and functional features upon senescence implementation. This phenotype can be transmitted in a paracrine manner, most likely through soluble secreted factors. A dynamic transcriptomic analysis during paracrine senescence confirmed the non-cell-autonomous transmission of this phenotype. Moreover, gene expression data combined with pharmacological and genetic manipulations of the major SASP signaling pathways suggest that the changes in myofibroblast phenotype are mainly mediated by the Notch/TGF-ß axis, involving a dynamic switch in the TGF-ß pathway. Our results reveal a novel link between senescence and myofibroblastic differentiation with potential implications in the physiological and pathological functions of myofibroblasts.
Assuntos
Senescência Celular , Miofibroblastos , Diferenciação Celular/fisiologia , Senescência Celular/genética , Fibroblastos/metabolismo , Miofibroblastos/metabolismo , Fenótipo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Rank signaling enhances stemness in mouse and human mammary epithelial cells (MECs) and mediates mammary tumor initiation. Mammary tumors initiated by oncogenes or carcinogen exposure display high levels of Rank and Rank pathway inhibitors have emerged as a new strategy for breast cancer prevention and treatment. Here, we show that ectopic Rank expression in the mammary epithelia unexpectedly delays tumor onset and reduces tumor incidence in the oncogene-driven Neu and PyMT models. Mechanistically, we have found that ectopic expression of Rank or exposure to Rankl induces senescence, even in the absence of other oncogenic mutations. Rank leads to DNA damage and senescence through p16/p19. Moreover, RANK-induced senescence is essential for Rank-driven stemness, and although initially translates into delayed tumor growth, eventually promotes tumor progression and metastasis. We uncover a dual role for Rank in the mammary epithelia: Rank induces senescence and stemness, delaying tumor initiation but increasing tumor aggressiveness.
Assuntos
Neoplasias da Mama/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Mamárias Animais/genética , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Envelhecimento/genética , Animais , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/genética , Dano ao DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Experimentais , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at lysine 4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus of the five ING proteins. ING5 facilitates histone H3 acetylation by the HBO1 complex, and also H4 acetylation by the MOZ/MORF complex. We show that ING5 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING5, which contains the nuclear localization sequence, is flexible and disordered, but it binds dsDNA with micromolar affinity. NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule, and they bind H3K4me3 in the same way as the isolated PHD. We have observed that ING5 can form heterodimers with the highly homologous ING4, and that two of three primary tumor-associated mutants in the N-terminal domain strongly destabilize the coiled-coil structure. They also affect cell proliferation and cell cycle phase distribution, suggesting a driver role in cancer progression.
Assuntos
Histonas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Histonas/química , Humanos , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Alinhamento de Sequência , Fatores de Transcrição/química , Proteínas Supressoras de Tumor/químicaRESUMO
Six1 is a developmental transcriptional regulator frequently overexpressed in human tumors. Recent results show that SIX1 also acts as a repressor of cell senescence, an antiproliferative response with a key role in tumor suppression, among other physiological and pathological settings. Here, we set to study the impact of SIX1 gain of function in transformation and tumorigenesis of fibroblasts, in connection with senescence. Using transcriptomic, histological, and functional analyses in murine tumors and cells of fibroblast origin, we show that SIX1 has a strong pro-tumorigenic action in this model, linked to the repression of a senescence-related gene signature and the induction of an undifferentiated phenotype mediated, at least in part, by the regulation of the stemness factor Sox2. Moreover, functional analyses with human glioma cell lines also show that SIX1 controls SOX2 expression, senescence and self-renewal in this model. Collectively, our results support a general link of SIX1 with senescence and SOX2-mediated cell plasticity in tumors.
Assuntos
Neoplasias Encefálicas/metabolismo , Carcinogênese/genética , Plasticidade Celular/genética , Senescência Celular/genética , Glioma/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Xenoenxertos , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Nus , Fatores de Transcrição SOXB1/genética , Transcriptoma , Transdução Genética , Carga Tumoral/genéticaRESUMO
The development of the inner ear complex cytoarchitecture and functional geometry requires the exquisite coordination of a variety of cellular processes in a temporal manner. At early stages of inner ear development several rounds of cell proliferation in the otocyst promote the growth of the structure. The apoptotic program is initiated in exceeding cells to adjust cell type numbers. Apoptotic cells are cleared by phagocytic cells that recognize the phosphatidylserine residues exposed in the cell membrane thanks to the energy supplied by autophagy. Specific molecular programs determine hair and supporting cell fate, these populations are responsible for the functions of the adult sensory organ: detection of sound, position and acceleration. The neurons that transmit auditory and balance information to the brain are also born at the otocyst by neurogenesis facilitated by autophagy. Cellular senescence participates in tissue repair, cancer and aging, situations in which cells enter a permanent cell cycle arrest and acquire a highly secretory phenotype that modulates their microenvironment. More recently, senescence has also been proposed to take place during vertebrate development in a limited number of transitory structures and organs; among the later, the endolymphatic duct in the inner ear. Here, we review these cellular processes during the early development of the inner ear, focusing on how the most recently described cellular senescence participates and cooperates with proliferation, apoptosis and autophagy to achieve otic morphogenesis and differentiation.
Assuntos
Orelha Interna/embriologia , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Orelha Interna/citologia , Orelha Interna/fisiologia , Humanos , Camundongos , Modelos Biológicos , Morfogênese/fisiologiaRESUMO
RhoE is a small GTPase involved in the regulation of actin cytoskeleton dynamics, cell cycle and apoptosis. The role of RhoE in cancer is currently controversial, with reports of both oncogenic and tumor-suppressive functions for RhoE. Using RhoE-deficient mice, we show here that the absence of RhoE blunts contact-inhibition of growth by inhibiting p27Kip1 nuclear translocation and cooperates in oncogenic transformation of mouse primary fibroblasts. Heterozygous RhoE+/gt mice are more susceptible to chemically induced skin tumors and RhoE knock-down results in increased metastatic potential of cancer cells. These results indicate that RhoE plays a role in suppressing tumor initiation and progression.
Assuntos
Transformação Celular Neoplásica/metabolismo , Inibição de Contato/fisiologia , Neoplasias Experimentais/patologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Western Blotting , Transformação Celular Neoplásica/patologia , Progressão da Doença , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Nus , Neoplasias Experimentais/metabolismoRESUMO
Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma.
Assuntos
Proteínas do Olho/genética , Melanoma/genética , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fatores de Crescimento Neural/genética , Serpinas/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Senescência Celular/genética , Progressão da Doença , Epistasia Genética , Proteínas do Olho/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Metástase Neoplásica , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismoAssuntos
Transformação Celular Neoplásica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteína 1 Inibidora do Crescimento , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Knockout , Metástase NeoplásicaRESUMO
The regulation of gene expression by microRNAs (miRNAs) is critical for normal development and physiology. Conversely, miRNA function is frequently impaired in cancer, and other pathologies, either by aberrant expression of individual miRNAs or dysregulation of miRNA synthesis. Here, we have investigated the impact of global disruption of miRNA biogenesis in primary fibroblasts of human or murine origin, through the knockdown of DGCR8, an essential mediator of the synthesis of canonical miRNAs. We find that the inactivation of DGCR8 in these cells results in a dramatic antiproliferative response, with the acquisition of a senescent phenotype. Senescence triggered by DGCR8 loss is accompanied by the upregulation of the cell-cycle inhibitor p21CIP1. We further show that a subset of senescence-associated miRNAs with the potential to target p21CIP1 is downregulated during DGCR8-mediated senescence. Interestingly, the antiproliferative response to miRNA biogenesis disruption is retained in human tumor cells, irrespective of p53 status. In summary, our results show that defective synthesis of canonical microRNAs results in cell-cycle arrest and cellular senescence in primary fibroblasts mediated by specific miRNAs, and thus identify global miRNA disruption as a novel senescence trigger.
Assuntos
Fibroblastos/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , Proteínas/metabolismo , Processos de Crescimento Celular/fisiologia , Senescência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/genética , Fibroblastos/citologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Ligação a RNA , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
ING proteins are tumor suppressors involved in the regulation of gene transcription, cell cycle arrest, apoptosis, and senescence. Here, we show that ING1b expression is upregulated by several DNA-damaging agents, in a p53-independent manner. ING1b stimulates DNA repair of a variety of DNA lesions requiring activation of multiple DNA repair pathways. Moreover, Ing1(-/-) cells showed impaired genomic DNA repair after H2O2 and neocarzinostatin treatment and this defect was reverted by overexpression of ING1b. Two tumor-derived ING1 mutants failed to promote DNA repair highlighting the physiological importance of the integrity of the PHD domain for ING1b DNA repair activity and suggesting a role in the prevention of tumor progression. Ing(-/-) cells showed higher basal levels of γ-H2AX and, upon DNA damage, γ-H2AX increase was greater and with faster kinetics compared to wild-type cells. Chromatin relaxation by Trichostatin A led to an exacerbated damage signal in both types of cells, but this effect was dependent on Ing1 status, and more pronounced in wild-type cells. Our results suggest that ING1 acts at early stages of the DNA damage response activating a variety of repair mechanisms and that this function of ING1 is targeted in tumors.
Assuntos
Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Expressão Gênica , Genoma Humano , Instabilidade Genômica , Histonas/metabolismo , Humanos , Proteína 1 Inibidora do Crescimento , Camundongos , Mutação de Sentido Incorreto , Isoformas de Proteínas/fisiologia , Proteínas Quinases/metabolismo , Regulação para CimaRESUMO
The protein ING4 binds to histone H3 trimethylated at Lys-4 (H3K4me3) through its C-terminal plant homeodomain, thus recruiting the HBO1 histone acetyltransferase complex to target promoters. The structure of the plant homeodomain finger bound to an H3K4me3 peptide has been described, as well as the disorder and flexibility in the ING4 central region. We report the crystal structure of the ING4 N-terminal domain, which shows an antiparallel coiled-coil homodimer with each protomer folded into a helix-loop-helix structure. This arrangement suggests that ING4 can bind simultaneously two histone tails on the same or different nucleosomes. Dimerization has a direct impact on ING4 tumor suppressor activity because monomeric mutants lose the ability to induce apoptosis after genotoxic stress. Homology modeling based on the ING4 structure suggests that other ING dimers may also exist.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Multimerização Proteica , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Cristalografia por Raios X , Histona Acetiltransferases/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Supressoras de Tumor/genéticaRESUMO
Cellular senescence is an effective tumor-suppressive mechanism that causes a stable proliferative arrest in cells with potentially oncogenic alterations. Here, we have investigated the role of the p33ING1 tumor suppressor in the regulation of cellular senescence in human primary fibroblasts. We show that p33ING1 triggers a senescent phenotype in a p53-dependent fashion. Also, endogenous p33ING1 protein accumulates in chromatin in oncogene-senescent fibroblasts and its silencing by RNA interference impairs senescence triggered by oncogenes. Notably, the ability to induce senescence is lost in a mutant version of p33ING1 present in human tumors. Using specific point mutants, we further show that recognition of the chromatin mark H3K4me3 is essential for induction of senescence by p33ING1. Finally, we demonstrate that ING1-induced senescence is associated to a specific genetic signature with a strong representation of chemokine and cytokine signaling factors, which significantly overlaps with that of oncogene-induced senescence. In summary, our results identify ING1 as a critical epigenetic regulator of cellular senescence in human fibroblasts and highlight its role in control of gene expression in the context of this tumor-protective response.
Assuntos
Senescência Celular/genética , Cromatina/metabolismo , Epigenômica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Quimiocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Genes Reguladores/fisiologia , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Análise em Microsséries , Proteínas Nucleares/metabolismo , Mutação Puntual , Ligação Proteica/genética , Interferência de RNA , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Inhibitor of growth 4 (ING4) is a member of the ING family of tumor suppressor proteins. In this study, we have analyzed the impact of two mutations in ING4 associated with human tumors (Y121N and N214D), testing their behavior in a series of functional, biochemical and structural analyses. We report that the N214D mutation dramatically dampened the ability of ING4 to inhibit proliferation, anchorage-independent growth or cell migration or to sensitize to cell death. In turn, the Y121N mutant did not differ significantly from wild-type ING4 in our assays. Neither of the mutations altered the normal subcellular localization of ING4, showing predominantly nuclear accumulation. We investigated the molecular basis of the defect in the activity of the N214D mutant. The folding and ability to bind histone marks of ING4 was not significantly altered by this mutation. Instead, we found that the functional impairment of the N214D mutant correlates with reduced protein stability due to increased proteasome-mediated degradation. In summary, our data demonstrates that a point mutation of ING4 associated to human tumors leads to the loss of several essential functions of ING4 pertinent to tumor protection and highlight the importance of ING4 function to prevent tumorigenesis.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/genética , Mutação/genética , Sarcoma/genética , Proteínas Supressoras de Tumor/genética , Animais , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Conformação Proteica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma/metabolismo , Sarcoma/patologia , Frações SubcelularesRESUMO
The ING family of tumor suppressor proteins controls several cellular functions relevant to antitumor protection, such as cell cycle control, apoptosis, senescence, or migration. ING proteins are functionally linked to the p53 pathway, and they participate in transcriptional control via the recognition of histone marks and recruitment of protein complexes with chromatin-modifying activity to specific promoters. Here, we have investigated the global effect of ING1 in gene regulation through genome-wide analysis of expression profiles in primary embryonic fibroblasts deficient for the Ing1 locus. We find that Ing1 has a predominant role as transcriptional repressor in this setting, affecting the expression of genes involved in a variety of cellular functions. Within the subset of genes showing differential expression, we have identified DGCR8, a protein involved in the early steps of microRNA biogenesis. We show that ING1 binds to the DGCR8 promoter and controls its transcription through chromatin regulation. We also find that ING1 and DGCR8 can cooperate in restraining proliferation. In summary, this study reveals a novel connection between ING1 and a regulator of microRNA biogenesis and identifies new links between tumor suppressor proteins and the microRNA machinery.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/biossíntese , Proteínas Nucleares/biossíntese , Proteínas/genética , Proteínas Supressoras de Tumor/biossíntese , Animais , Western Blotting , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , MicroRNAs/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA , Transcrição Gênica , Proteínas Supressoras de Tumor/genéticaRESUMO
The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at position K4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus in the five members of the ING family. ING4 facilitates histone H3 acetylation by the HBO1 complex. Here, we show that ING4 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING4, which contains the nuclear localization sequence, is disordered and flexible and does not directly interact with p53, or does it with very low affinity, in contrast to previous findings. The NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule. The detailed NMR analysis of the full-length dimeric protein binding to histone H3K4me3 shows essentially the same binding site and affinity as the isolated PHD finger. Therefore, the ING4 dimer has two identical and independent binding sites for H3K4me3 tails, which, in the context of the chromatin, could belong to the same or to different nucleosomes. These results show that ING4 is a bivalent reader of the chromatin H3K4me3 modification and suggest a mechanism for enhanced targeting of the HBO1 complex to specific chromatin sites. This mechanism could be common to other ING-containing remodeling complexes.