RESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease observed with aging that represents the most common form of dementia. To date, therapies targeting end-stage disease plaques, tangles, or inflammation have limited efficacy. Therefore, we set out to identify a potential earlier targetable phenotype. Utilizing a mouse model of AD and human fetal cells harboring mutant amyloid precursor protein, we show cell intrinsic neural precursor cell (NPC) dysfunction precedes widespread inflammation and amyloid plaque pathology, making it the earliest defect in the evolution of the disease. We demonstrate that reversing impaired NPC self-renewal via genetic reduction of USP16, a histone modifier and critical physiological antagonist of the Polycomb Repressor Complex 1, can prevent downstream cognitive defects and decrease astrogliosis in vivo. Reduction of USP16 led to decreased expression of senescence gene Cdkn2a and mitigated aberrant regulation of the Bone Morphogenetic Signaling (BMP) pathway, a previously unknown function of USP16. Thus, we reveal USP16 as a novel target in an AD model that can both ameliorate the NPC defect and rescue memory and learning through its regulation of both Cdkn2a and BMP signaling.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Senescência Celular , Modelos Animais de Doenças , Inflamação , Camundongos , Camundongos Transgênicos , Placa Amiloide , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
SMAD pathways govern epithelial proliferation, and transforming growth factor ß (TGF-ß and BMP signaling through SMAD members has distinct effects on mammary development and homeostasis. Here, we show that LEFTY1, a secreted inhibitor of NODAL/SMAD2 signaling, is produced by mammary progenitor cells and, concomitantly, suppresses SMAD2 and SMAD5 signaling to promote long-term proliferation of normal and malignant mammary epithelial cells. In contrast, BMP7, a NODAL antagonist with context-dependent functions, is produced by basal cells and restrains progenitor cell proliferation. In normal mouse epithelium, LEFTY1 expression in a subset of luminal cells and rare basal cells opposes BMP7 to promote ductal branching. LEFTY1 binds BMPR2 to suppress BMP7-induced activation of SMAD5, and this LEFTY1-BMPR2 interaction is specific to tumor-initiating cells in triple-negative breast cancer xenografts that rely on LEFTY1 for growth. These results suggest that LEFTY1 is an endogenous dual-SMAD inhibitor and that suppressing its function may represent a therapeutic vulnerability in breast cancer.
Assuntos
Transdução de Sinais , Fator de Crescimento Transformador beta , Animais , Carcinogênese , Transformação Celular Neoplásica , CamundongosRESUMO
Regulation of the Wnt pathway in stem cells and primary tissues is still poorly understood. Here we report that Usp16, a negative regulator of Bmi1/PRC1 function, modulates the Wnt pathway in mammary epithelia, primary human fibroblasts and MEFs, affecting their expansion and self-renewal potential. In mammary glands, reduced levels of Usp16 increase tissue responsiveness to Wnt, resulting in upregulation of the downstream Wnt target Axin2, expansion of the basal compartment and increased in vitro and in vivo epithelial regeneration. Usp16 regulation of the Wnt pathway in mouse and human tissues is at least in part mediated by activation of Cdkn2a, a regulator of senescence. At the molecular level, Usp16 affects Rspo-mediated phosphorylation of LRP6. In Down's Syndrome (DS), triplication of Usp16 dampens the activation of the Wnt pathway. Usp16 copy number normalization restores normal Wnt activation in Ts65Dn mice models. Genetic upregulation of the Wnt pathway in Ts65Dn mice rescues the proliferation defect observed in mammary epithelial cells. All together, these findings link important stem cell regulators like Bmi1/Usp16 and Cdkn2a to Wnt signaling, and have implications for designing therapies for conditions, like DS, aging or degenerative diseases, where the Wnt pathway is hampered.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação da Expressão Gênica , Ubiquitina Tiolesterase/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Ubiquitina Tiolesterase/genética , Proteína Wnt3A/metabolismoRESUMO
Down's syndrome results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene-dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, which are trisomic for 132 genes homologous to genes on human chromosome 21, triplication of Usp16 reduces the self-renewal of haematopoietic stem cells and the expansion of mammary epithelial cells, neural progenitors and fibroblasts. In addition, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from histone H2A on lysine 119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal Usp16 allele or by short interfering RNAs, largely rescues all of these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and postnatal neural progenitors, whereas downregulation of USP16 partially rescues the proliferation defects of Down's syndrome fibroblasts. Taken together, these results suggest that USP16 has an important role in antagonizing the self-renewal and/or senescence pathways in Down's syndrome and could serve as an attractive target to ameliorate some of the associated pathologies.
Assuntos
Síndrome de Down/metabolismo , Síndrome de Down/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Ubiquitina Tiolesterase/metabolismo , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Animais , Proliferação de Células , Senescência Celular , Cromossomos Humanos Par 21/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Síndrome de Down/genética , Epitélio/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Dosagem de Genes , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/patologia , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Terapia de Alvo Molecular , Trissomia/genética , Ubiquitina Tiolesterase/genética , UbiquitinaçãoRESUMO
CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.
Assuntos
Antígenos de Diferenciação/metabolismo , Antígeno CD47/imunologia , Neoplasias/imunologia , RNA Mensageiro/genética , Receptores Imunológicos/metabolismo , Anticorpos/imunologia , Antígeno CD47/genética , Divisão Celular/imunologia , Citometria de Fluxo , Humanos , Neoplasias/patologia , Neoplasias/terapia , Fagocitose/imunologia , Prognóstico , Análise de SobrevidaRESUMO
MicroRNAs profoundly impact hematopoietic cells by regulating progenitor cell-fate decisions, as well as mature immune effector function. However to date, microRNAs that regulate hematopoietic stem cell (HSC) function have been less well characterized. Here we show that microRNA-125b (miR-125b) is highly expressed in HSCs and its expression decreases in committed progenitors. Overexpression of miR-125b in mouse HSC enhances their function, demonstrated through serial transplantation of highly purified HSC, and enriches for the previously described Slamf1(lo)CD34(-) lymphoid-balanced and the Slamf1(neg)CD34(-) lymphoid-biased cell subsets within the multipotent HSC (CD34-KLS) fraction. Mature peripheral blood cells derived from the miR-125b-overexpressing HSC are skewed toward the lymphoid lineage. Consistent with this observation, miR-125b overexpression significantly increases the number of early B-progenitor cells within the spleen and induces the expansion and enrichment of the lymphoid-balanced and lymphoid-biased HSC subset via an antiapoptotic mechanism, reducing the mRNA expression levels of two proapoptotic targets, Bmf and KLF13. The antiapoptotic effect of miR-125b is more pronounced in the lymphoid-biased HSC subset because of their intrinsic higher baseline levels of apoptosis. These effects of miR-125b are associated with the development of lymphoproliferative disease, marked by expansion of CD8(+) T lymphocytes. Taken together, these data reveal that miR-125b regulates HSC survival and can promote lymphoid-fate decisions at the level of the HSC by preferentially expanding lymphoid-balanced and lymphoid-biased HSC.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Subpopulações de Linfócitos , Linfócitos/fisiologia , MicroRNAs/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD34/genética , Antígenos CD34/metabolismo , Apoptose , Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Análise em Microsséries , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Transplante de Células-Tronco , Quimeras de TransplanteRESUMO
To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.
Assuntos
Neoplasias da Mama/patologia , Metástase Neoplásica , Células-Tronco Neoplásicas/citologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de NeoplasiasRESUMO
TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.
Assuntos
Metástase Neoplásica , Proteínas Smad/metabolismo , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Mutação , Transplante de Neoplasias , Organismos Livres de Patógenos Específicos , Fatores de Transcrição , Proteína Supressora de Tumor p53/genética , Proteínas ras/metabolismoRESUMO
The assembly of the Smad complex is critical for TGFbeta signaling, yet the mechanisms that inactivate or empower nuclear Smad complexes are less understood. By means of siRNA screen we identified FAM (USP9x), a deubiquitinase acting as essential and evolutionarily conserved component in TGFbeta and bone morphogenetic protein signaling. Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits Smad4 by impeding association with phospho-Smad2. FAM reverts this negative modification, re-empowering Smad4 function. FAM opposes the activity of Ectodermin/Tif1gamma (Ecto), a nuclear factor for which we now clarify a prominent role as Smad4 monoubiquitin ligase. Our study points to Smad4 monoubiquitination and deubiquitination as a way for cells to set their TGFbeta responsiveness: loss of FAM disables Smad4-dependent responses in several model systems, with Ecto being epistatic to FAM. This defines a regulative ubiquitination step controlling Smads that is parallel to those impinging on R-Smad phosphorylation.
Assuntos
Proteína Smad4/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular Tumoral , Embrião não Mamífero/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação , XenopusRESUMO
MicroRNAs are crucial modulators of gene expression, yet their involvement as effectors of growth factor signalling is largely unknown. Ligands of the transforming growth factor-beta superfamily are essential for development and adult tissue homeostasis. In early Xenopus embryos, signalling by the transforming growth factor-beta ligand Nodal is crucial for the dorsal induction of the Spemann's organizer. Here we report that Xenopus laevis microRNAs miR-15 and miR-16 restrict the size of the organizer by targeting the Nodal type II receptor Acvr2a. Endogenous miR-15 and miR-16 are ventrally enriched as they are negatively regulated by the dorsal Wnt/beta-catenin pathway. These findings exemplify the relevance of microRNAs as regulators of early embryonic patterning acting at the crossroads of fundamental signalling cascades.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Receptores de Activinas Tipo II/biossíntese , Receptores de Activinas Tipo II/genética , Ativinas/metabolismo , Animais , Padronização Corporal , MicroRNAs/genética , Proteína Nodal , Organizadores Embrionários/embriologia , Organizadores Embrionários/metabolismo , Proteínas Wnt/metabolismo , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMO
During development and tissue homeostasis, cells must integrate different signals. We investigated how cell behavior is controlled by the combined activity of transforming growth factor-beta (TGF-beta) and receptor tyrosine kinase (RTK) signaling, whose integration mechanism is unknown. We find that RTK/Ras/MAPK (mitogen-activated protein kinase) activity induces p53 N-terminal phosphorylation, enabling the interaction of p53 with the TGF-beta-activated Smads. This mechanism confines mesoderm specification in Xenopus embryos and promotes TGF-beta cytostasis in human cells. These data indicate a mechanism to allow extracellular cues to specify the TGF-beta gene-expression program.
Assuntos
Proliferação de Células , Embrião não Mamífero/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/metabolismo , Substituição de Aminoácidos , Animais , Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Idelta/metabolismo , Linhagem Celular Tumoral , Desenvolvimento Embrionário , Indução Embrionária , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesoderma/metabolismo , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Smad/metabolismo , XenopusRESUMO
TGF-beta proteins are main regulators of blood vessel development and maintenance. Here, we report an unprecedented link between TGF-beta signaling and arterial hypertension based on the analysis of mice mutant for Emilin1, a cysteine-rich secreted glycoprotein expressed in the vascular tree. Emilin1 knockout animals display increased blood pressure, increased peripheral vascular resistance, and reduced vessel size. Mechanistically, we found that Emilin1 inhibits TGF-beta signaling by binding specifically to the proTGF-beta precursor and preventing its maturation by furin convertases in the extracellular space. In support of these findings, genetic inactivation of Emilin1 causes increased TGF-beta signaling in the vascular wall. Strikingly, high blood pressure observed in Emilin1 mutants is rescued to normal levels upon inactivation of a single TGF-beta1 allele. This study highlights the importance of modulation of TGF-beta availability in the pathogenesis of hypertension.
Assuntos
Pressão Sanguínea/fisiologia , Homeostase , Glicoproteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Artérias/citologia , Artérias/metabolismo , Furina/metabolismo , Dosagem de Genes , Genes Reporter , Humanos , Hipertensão/etiologia , Hipertensão/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína Nodal , Fenótipo , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
TGF-beta signaling is essential for development and proliferative homeostasis. During embryogenesis, maternal determinants act in concert with TGF-beta signals to form mesoderm and endoderm. In contrast, ectoderm specification requires the TGF-beta response to be attenuated, although the mechanisms by which this is achieved remain unknown. In a functional screen for ectoderm determinants, we have identified Ectodermin (Ecto). In Xenopus embryos, Ecto is essential for the specification of the ectoderm and acts by restricting the mesoderm-inducing activity of TGF-beta signals to the mesoderm and favoring neural induction. Ecto is a RING-type ubiquitin ligase for Smad4, a TGF-beta signal transducer. Depletion of Ecto in human cells enforces TGF-beta-induced cytostasis and, moreover, plays a causal role in limiting the antimitogenic effects of Smad4 in tumor cells. We propose that Ectodermin is a key switch in the control of TGF-beta gene responses during early embryonic development and cell proliferation.
Assuntos
Blástula/metabolismo , Núcleo Celular/metabolismo , Camadas Germinativas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Sequência de Bases , Blástula/citologia , Receptores de Proteínas Morfogenéticas Ósseas , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Colo/citologia , Colo/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Camadas Germinativas/citologia , Humanos , Dados de Sequência Molecular , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad4 , Transativadores/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Xenopus/embriologia , Proteínas de Xenopus/genéticaRESUMO
p53 is a protein with many talents. One of the most fundamental is the ability to act as essential growth checkpoint that protects cells against cellular transformation. p53 does so through the induction of genes leading to growth arrest or apoptosis. Most of the studies focusing on the mechanisms of p53 activity have been performed in cultured cells upon treatment with well-established p53-activating inputs, such as high doses of radiations, DNA-damaging drugs and activated oncogenes. However, how the tumor suppressive functions of p53 become concerted with the extracellular cues arriving at the cell surface during tissue homeostasis, remains largely unknown. Intriguingly, two recent papers have shed new light into this unexplored field, indicating that p53 plays a key role in TGF-beta-induced growth arrest and, unexpectedly, in the developmental effects of TGF-beta in early embryos. Here we review and comment on these findings and on their implications for cancer biology.