RESUMO
Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.
Assuntos
Técnicas Biossensoriais , Proteínas Proto-Oncogênicas c-akt , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas Biossensoriais/métodosRESUMO
Interleukin-7 (IL-7) supports the growth and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL), particularly the early T-cell precursor subtype (ETP-ALL), which frequently has activating mutations of IL-7 signaling. Signal transducer and activator of transcription (STAT5) is an attractive therapeutic target because it is almost universally activated in ETP-ALL, even in the absence of mutations of upstream activators such as the IL-7 receptor (IL-7R), Janus kinase, and Fms-like tyrosine kinase 3 (FLT3). To examine the role of activated STAT5 in ETP-ALL, we have used a Lmo2-transgenic (Lmo2Tg) mouse model in which we can monitor chemoresistant preleukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs) that drive T-ALL development and relapse following chemotherapy. Using IL-7R-deficient Lmo2Tg mice, we show that IL-7 signaling was not required for the formation of pre-LSCs but essential for their expansion and clonal evolution into LSCs to generate T-ALL. Activated STAT5B was sufficient for the development of T-ALL in IL-7R-deficient Lmo2Tg mice, indicating that inhibition of STAT5 is required to block the supportive signals provided by IL-7. To further understand the role of activated STAT5 in LSCs of ETP-ALL, we developed a new transgenic mouse that enables T-cell specific and doxycycline-inducible expression of the constitutively activated STAT5B1∗6 mutant. Expression of STAT5B1∗6 in T cells had no effect alone but promoted expansion and chemoresistance of LSCs in Lmo2Tg mice. Pharmacologic inhibition of STAT5 with pimozide-induced differentiation and loss of LSCs, while enhancing response to chemotherapy. Furthermore, pimozide significantly reduced leukemia burden in vivo and overcame chemoresistance of patient-derived ETP-ALL xenografts. Overall, our results demonstrate that STAT5 is an attractive therapeutic target for eradicating LSCs in ETP-ALL.
Assuntos
Células Precursoras de Linfócitos T , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Camundongos , Animais , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Interleucina-7/genética , Interleucina-7/metabolismo , Pimozida/uso terapêutico , Camundongos TransgênicosRESUMO
ETP-ALL (Early T cell Progenitor Acute Lymphoblastic Leukemia) represents a high-risk subtype of T cell acute lymphocytic leukemia (T-ALL). Therapeutically, ETP-ALL patients frequently relapse after conventional chemotherapy highlighting the need for alternative therapeutic approaches. Using our ZEB2Tg ETP-ALL mouse model we previously documented the potential utility of the catalytic LSD1 inhibitor (GSK2879552) for treating mouse/human ETP-ALL. However, this approach proved to be inefficient, especially in killing human LOUCY cell ETP-ALL xenografts in vivo. Here we have revealed the novel involvement of ZEB2/LSD1 complexes in repressing the intrinsic apoptosis pathway by inhibiting the expression of several pro-apoptotic proteins such as BIM (BCL2L11) as a major driver for ETP-ALL survival. Treatment with LSD1i (particularly with the steric inhibitor SP2509) restored the expression of ZEB2/LSD1 pro-apoptotic BIM (BCL2L11) target. In combination with a JAK/STAT pathway inhibitor (JAKi, Ruxolitinib) or with a direct inhibitor of the anti-apoptotic BCL2 protein (BCL2i, ABT-199) resistance of human and mouse ETP-ALL to LSD1i was reversed. This new combination approach efficiently inhibited the growth of human and mouse ETP-ALL cells in vivo by enhancing their differentiation and triggering an apoptotic response. These results set the stage for novel combination therapies to be used in clinical trials to treat ETP-ALL patients.
Assuntos
Inibidores de Janus Quinases , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Camundongos , Animais , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteína 11 Semelhante a Bcl-2/metabolismo , Inibidores de Janus Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Histona Desmetilases/metabolismoRESUMO
Cancer cell growth is dependent upon the sustainability of proliferative signaling and resisting cell death. Macroautophagy/autophagy promotes cancer cell growth by providing nutrients to cells and preventing cell death. This is in contrast to autophagy promoting cell death under some conditions. The mechanism regulating autophagy-mediated cancer cell growth remains unclear. Herein, we demonstrate that TSSC4 (tumor suppressing subtransferable candidate 4) is a novel tumor suppressor that suppresses cancer cell growth and tumor growth and prevents cell death induction during excessive growth by inhibiting autophagy. The oncogenic proteins ERBB2 (erb-b2 receptor tyrosine kinase 2) and the activation EGFR mutant (EGFRvIII, epidermal growth factor receptor variant III) promote cell growth and TSSC4 expression in breast cancer and glioblastoma multiforme (GBM) cells, respectively. In EGFRvIII-expressing GBM cells, TSSC4 knockout shifted the function of autophagy from a pro-cell survival role to a pro-cell death role during prolonged cell growth. Furthermore, the interaction of TSSC4 with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) via its conserved LC3-interacting region (LIR) contributes to its inhibition of autophagy. Finally, TSSC4 suppresses tumorsphere formation and tumor growth by inhibiting autophagy and maintaining cell survival in tumorspheres. Taken together, sustainable cancer cell growth can be achieved by autophagy inhibition via TSSC4 expression.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; CQ: chloroquine; EGFRvIII: epidermal growth factor receptor variant III; ERBB2: erb-b2 receptor tyrosine kinase 2; GBM: glioblastoma multiforme; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule Associated protein 1 light chain 3; TSSC4: tumor suppressing subtransferable candidate 4.
Assuntos
Autofagia , Glioblastoma , Transformação Celular Neoplásica , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Receptor ErbB-2 , Proteínas Supressoras de TumorRESUMO
The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]âMPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.
Assuntos
Linhagem da Célula , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Células da Medula Óssea/patologia , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Transgênicos , RNA-Seq , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.
Assuntos
Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal/genética , Histona Desmetilases/metabolismo , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição da Família Snail/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células HEK293 , Células HL-60 , Histona Desmetilases/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Ligação Proteica , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismoRESUMO
Cancer is a major and still increasing cause of death in humans. Most cancer cells have a fundamentally different metabolic profile from that of normal tissue. This shift away from mitochondrial ATP synthesis via oxidative phosphorylation towards a high rate of glycolysis, termed Warburg effect, has long been recognized as a paradigmatic hallmark of cancer, supporting the increased biosynthetic demands of tumor cells. Here we show that deletion of apoptosis-inducing factor (AIF) in a KrasG12D-driven mouse lung cancer model resulted in a marked survival advantage, with delayed tumor onset and decreased malignant progression. Mechanistically, Aif deletion leads to oxidative phosphorylation (OXPHOS) deficiency and a switch in cellular metabolism towards glycolysis in non-transformed pneumocytes and at early stages of tumor development. Paradoxically, although Aif-deficient cells exhibited a metabolic Warburg profile, this bioenergetic change resulted in a growth disadvantage of KrasG12D-driven as well as Kras wild-type lung cancer cells. Cell-autonomous re-expression of both wild-type and mutant AIF (displaying an intact mitochondrial, but abrogated apoptotic function) in Aif-knockout KrasG12D mice restored OXPHOS and reduced animal survival to the same level as AIF wild-type mice. In patients with non-small cell lung cancer, high AIF expression was associated with poor prognosis. These data show that AIF-regulated mitochondrial respiration and OXPHOS drive the progression of lung cancer.
Assuntos
Fator de Indução de Apoptose/fisiologia , Carcinogênese/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Progressão da Doença , Glicólise , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação OxidativaRESUMO
ZEB1 and ZEB2 are structurally related E-box binding homeobox transcription factors that induce epithelial to mesenchymal transitions during development and disease. As such, they regulate cancer cell invasion, dissemination and metastasis of solid tumors. In addition, their expression is associated with the gain of cancer stem cell properties and resistance to therapy. Using conditional loss-of-function mice, we previously demonstrated that Zeb2 also plays pivotal roles in hematopoiesis, controlling important cell fate decisions, lineage commitment and fidelity. In addition, upon Zeb2 overexpression, mice spontaneously develop immature T-cell lymphoblastic leukemia. Here we show that pre-leukemic Zeb2-overexpressing thymocytes are characterized by a differentiation delay at beta-selection due to aberrant activation of the interleukin-7 receptor signaling pathway. Notably, and in contrast to Lmo2-overexpressing thymocytes, these pre-leukemic Zeb2-overexpressing T-cell progenitors display no acquired self-renewal properties. Finally, Zeb2 activation in more differentiated T-cell precursor cells can also drive malignant T-cell development, suggesting that the early T-cell differentiation delay is not essential for Zeb2-mediated leukemic transformation. Altogether, our data suggest that Zeb2 and Lmo2 drive malignant transformation of immature T-cell progenitors via distinct molecular mechanisms.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Transformação Celular Neoplásica/genética , Proteínas com Domínio LIM/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica , Hematopoese , Humanos , Imuno-Histoquímica , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Proteínas com Domínio LIM/metabolismo , Camundongos , Gradação de Tumores , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Timo/patologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismoRESUMO
Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.
Assuntos
Benzoatos/farmacologia , Ciclopropanos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Repressoras/metabolismo , Animais , Benzoatos/uso terapêutico , Linhagem Celular Tumoral , Ciclopropanos/uso terapêutico , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Repressoras/genética , Regulação para Cima , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
Snail family members regulate epithelial-to-mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation-induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.
Assuntos
Mucosa Intestinal/metabolismo , Intestinos/citologia , Fatores de Transcrição/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/genética , Leucemia de Células T/fisiopatologia , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Animais , Western Blotting , Imunoprecipitação da Cromatina , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Técnicas Histológicas , Proteínas de Homeodomínio/imunologia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Janus Quinases/metabolismo , Estimativa de Kaplan-Meier , Cariotipagem , Luciferases , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-7/metabolismo , Proteínas Repressoras/imunologia , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.
Assuntos
Endotélio Vascular/metabolismo , Hemangiossarcoma/metabolismo , Proteína Supressora de Tumor p53/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Hemangiossarcoma/patologia , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Epithelial homeostasis within the epidermis is maintained by means of multiple cell-cell adhesion complexes such as adherens junctions, tight junctions, gap junctions, and desmosomes. These complexes co-operate in the formation and the regulation of the epidermal barrier. Disruption of the epidermal barrier through the deregulation of the above complexes is the cause behind a number of skin disorders such as psoriasis, dermatitis, keratosis, and others. During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their adhesive capacities and gain mesenchymal properties. ZEB transcription factors are key inducers of EMT. In order to gain a better understanding of the functional role of ZEB2 in epidermal homeostasis, we generated a mouse model with conditional overexpression of Zeb2 in the epidermis. Our analysis revealed that Zeb2 expression in the epidermis leads to hyperproliferation due to the combined downregulation of different tight junction proteins compromising the epidermal barrier. Using two epidermis-specific in vivo models and in vitro promoter assays, we identified occludin as a new Zeb2 target gene. Immunohistological analysis performed on human skin biopsies covering various pathogeneses revealed ZEB2 expression in the epidermis of pemphigus vulgaris. Collectively, our data support the notion for a potential role of ZEB2 in intracellular signaling of this disease.
Assuntos
Proteínas de Homeodomínio/fisiologia , Proteínas Repressoras/fisiologia , Pele/metabolismo , Junções Íntimas/metabolismo , Animais , Linhagem Celular , Transição Epitelial-Mesenquimal/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Ocludina/metabolismo , Pênfigo/genética , Pênfigo/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Pele/patologia , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
Current models of brain development support the view that VEGF, a signaling protein secreted by neuronal cells, regulates angiogenesis and neuronal development. Here we demonstrate an autonomous and pivotal role for endothelial cell-derived VEGF that has far-reaching consequences for mouse brain development. Selective deletion of Vegf from endothelial cells resulted in impaired angiogenesis and marked perturbation of cortical cytoarchitecture. Abnormal cell clusters or heterotopias were detected in the marginal zone, and disorganization of cortical cells induced several malformations, including aberrant cortical lamination. Critical events during brain development-neuronal proliferation, differentiation, and migration were significantly affected. In addition, axonal tracts in the telencephalon were severely defective in the absence of endothelial VEGF. The unique roles of endothelial VEGF cannot be compensated by neuronal VEGF and underscores the high functional significance of endothelial VEGF for cerebral cortex development and from disease perspectives.
Assuntos
Córtex Cerebral , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores Etários , Animais , Bromodesoxiuridina/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Antígeno Ki-67/metabolismo , Laminina/metabolismo , Lectinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Loss of the tumor suppressor PTEN is a common occurrence in prostate cancer. This aberration leads to the ectopic activation of the PI3K-Akt pathway, which promotes tumor growth. Here, we show that the transcription factor Gata3 is progressively lost in Pten-deficient mouse prostate tumors as a result of both transcriptional down-regulation and increased proteasomal degradation. To determine the significance of this loss, we used conditional loss- and gain-of-function approaches to manipulate Gata3 expression levels in prostate tumors. Our results show that Gata3 inactivation in Pten-deficient prostates accelerates tumor invasion. Conversely, enforced expression of GATA3 in Pten-deficient tissues markedly delays tumor progression. In Pten-deficient prostatic ducts, enforced GATA3 prevented Akt activation, which correlated with the down-regulation of Pik3cg and Pik3c2a mRNAs, encoding respectively class I and II PI3K subunits. Remarkably, the majority of human prostate tumors similarly show loss of active GATA3 as they progress to the aggressive castrate-resistant stage. In addition, GATA3 expression levels in hormone-sensitive tumors holds predictive value for tumor recurrence. Together, these data establish Gata3 as an important regulator of prostate cancer progression.
Assuntos
Fator de Transcrição GATA3/metabolismo , PTEN Fosfo-Hidrolase/deficiência , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Regulação para Baixo , Feminino , Fator de Transcrição GATA3/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Fetal growth plate cartilage is nonvascularized, and chondrocytes largely develop in hypoxic conditions. We previously found that mice lacking the hypoxia-inducible transcription factor HIF-1α in cartilage show massive death of centrally located, hypoxic chondrocytes. A similar phenotype was observed in mice with genetic ablation of either all or specifically the diffusible isoforms of vascular endothelial growth factor (VEGF), a prime angiogenic target of HIF-1α. Here, we assessed whether VEGF is a critical downstream component of the HIF-1α-dependent survival pathway in chondrocytes. We used a genetic approach to conditionally overexpress VEGF164 in chondrocytes lacking HIF-1α, evaluating potential rescuing effects. The effectiveness of the strategy was validated by showing that transgenic expression of VEGF164 in Col2-Cre;VEGF(f/f) mice stimulated angiogenesis in the perichondrium, fully corrected the excessive hypoxia of VEGF-deficient chondrocytes, and completely prevented chondrocyte death. Yet, similarly crossed double-mutant embryos lacking HIF-1α and overexpressing VEGF164 in the growth plate cartilage still displayed a central cell death phenotype, albeit slightly delayed and less severe compared with mice exclusively lacking HIF-1α. Transgenic VEGF164 induced massive angiogenesis in the perichondrium, yet this only partially relieved the aberrant hypoxia present in HIF-1α-deficient cartilage and thereby likely inflicted only a partial rescue effect. In fact, excessive hypoxia and failure to upregulate phosphoglycerate-kinase 1 (PGK1), a key enzyme of anaerobic glycolytic metabolism, were among the earliest manifestations of HIF-1α deficiency in cartilaginous bone templates, and reduced PGK1 expression was irrespective of transgenic VEGF164. These findings suggest that HIF-1α activates VEGF-independent cell-autonomous mechanisms to sustain oxygen levels in the challenged avascular cartilage by reducing oxygen consumption. Hence, regulation of the metabolic pathways by HIF-1α and VEGF-dependent regulation of angiogenesis coordinately act to maintain physiological cartilage oxygenation. We conclude that VEGF and HIF-1α are critical preservers of chondrocyte survival by ensuring an adequate balance between availability and handling of oxygen in developing growth cartilage.
Assuntos
Cartilagem/fisiologia , Sobrevivência Celular/fisiologia , Condrócitos/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Consumo de Oxigênio/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Apoptose , Cartilagem/citologia , Cartilagem/embriologia , Camundongos , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased ß1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult.
Assuntos
Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Transição Epitelial-Mesenquimal , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/fisiologia , Proteínas Repressoras/fisiologia , Animais , Caderinas/metabolismo , Movimento Celular , Feminino , Citometria de Fluxo , Genes Letais , Células-Tronco Hematopoéticas/metabolismo , Integrases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Dedos de ZincoRESUMO
Mdm2 and Mdm4 are critical negative regulators of p53. A large body of evidence indicates that elevated expression of either Mdm2 or Mdm4 may favor tumor formation by inhibiting p53 tumor suppression function. To explore this possibility in vivo, we generated conditional Mdm2 and Mdm4 transgenic mice. We show that although both transgenes are designed to be expressed ubiquitously and at comparable levels, only the Mdm4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasome-dependent manner, indicating that cells are equipped with efficient mechanisms that prevent Mdm2 accumulation in vivo. Mice that are homozygous for the Mdm4 transgene die during embryogenesis owing to severe vascular maturation defects. Importantly, this lethality is not rescued on a p53-null background, indicating that high levels of Mdm4 impact on a pathway(s) other than p53 that controls vascular and embryonic development. Mice expressing a single copy of the Mdm4 transgene are viable and, surprisingly, are not prone to spontaneous, radiation-induced or Eµ-myc-induced tumor formation. The findings have clear implications for cancer etiology as well as for cancer therapy.
Assuntos
Epitopos , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Estimativa de Kaplan-Meier , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/etiologia , Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Distribuição Tecidual , Transgenes , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
The AP-1 transcription factor c-Jun is essential for cellular proliferation in many cell types, but the molecular link between growth factors and c-Jun activation has been enigmatic. In this study we identify a previously uncharacterized RING-domain-containing protein, RACO-1 (RING domain AP-1 co-activator-1), as a c-Jun co-activator that is regulated by growth factor signalling. RACO-1 interacted with c-Jun independently of amino-terminal phosphorylation, and was both necessary and sufficient for c-Jun/AP-1 activation. Growth factor-mediated stimulation of AP-1 was attributable to MEK/ERK-dependent stabilization of RACO-1 protein. Stimulation of the MEK/ERK pathway strongly promoted Lys 63-linked ubiquitylation of RACO-1, which antagonized Lys 48-linked degradative auto-ubiquitylation of the same Lys residues. RACO-1 depletion reduced cellular proliferation and decreased expression of several growth-associated AP-1 target genes, such as cdc2, cyclinD1 and hb-egf. Moreover, transgenic overexpression of RACO-1 augmented intestinal tumour formation triggered by aberrant Wnt signalling and cooperated with oncogenic Ras in colonic hyperproliferation. Thus RACO-1 is a co-activator that links c-Jun to growth factor signalling and is essential for AP-1 function in proliferation.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Genes ras/genética , Humanos , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células NIH 3T3 , Fosforilação , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf(164) isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.