RESUMO
Nuclear pore complexes (NPCs) regulate nuclear-cytoplasmic transport, transcription, and genome integrity in eukaryotic cells. However, their functional roles in cancer remain poorly understood. We interrogated the evolutionary transcriptomic landscape of NPC components, nucleoporins (Nups), from primary to advanced metastatic human prostate cancer (PC). Focused loss-of-function genetic screen of top-upregulated Nups in aggressive PC models identified POM121 as a key contributor to PC aggressiveness. Mechanistically, POM121 promoted PC progression by enhancing importin-dependent nuclear transport of key oncogenic (E2F1, MYC) and PC-specific (AR-GATA2) transcription factors, uncovering a pharmacologically targetable axis that, when inhibited, decreased tumor growth, restored standard therapy efficacy, and improved survival in patient-derived pre-clinical models. Our studies molecularly establish a role of NPCs in PC progression and give a rationale for NPC-regulated nuclear import targeting as a therapeutic strategy for lethal PC. These findings may have implications for understanding how NPC deregulation contributes to the pathogenesis of other tumor types.
Assuntos
Fator de Transcrição E2F1/metabolismo , Glicoproteínas de Membrana/metabolismo , Poro Nuclear/fisiologia , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Carcinogênese , Núcleo Celular/metabolismo , Proliferação de Células , Fator de Transcrição GATA2/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Membrana Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares , Transdução de SinaisRESUMO
The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.
Assuntos
Medula Óssea/irrigação sanguínea , Microambiente Celular , Hematopoese , Células-Tronco Hematopoéticas , Análise de Célula Única , Nicho de Células-Tronco , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Linhagem da Célula , Endotélio Vascular/citologia , Feminino , Regulação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA-Seq , Receptores Notch/metabolismo , Nicho de Células-Tronco/genética , Estresse Fisiológico/genética , Transcriptoma/genéticaRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathological states, such as viral infections and certain cancers, coincide with ERV expression, suggesting that transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but the conserved nature of these elements has, until recently, rendered their expression profiling problematic. Here, we provide comprehensive HERV-K HML-2 expression profiles specific for productively HIV-1-infected primary human CD4+ T cells. We combined enrichment of HIV-1 infected cells using a reporter virus expressing a surface reporter for gentle and efficient purification with long-read single-molecule real-time sequencing. We show that three HML-2 proviruses-6q25.1, 8q24.3, and 19q13.42-are upregulated on average between 3- and 5-fold in HIV-1-infected CD4+ T cells. One provirus, HML-2 12q24.33, in contrast, was repressed in the presence of active HIV replication. In conclusion, this report identifies the HERV-K HML-2 loci whose expression profiles differ upon HIV-1 infection in primary human CD4+ T cells. These data will help pave the way for further studies on the influence of endogenous retroviruses on HIV-1 replication.IMPORTANCE Endogenous retroviruses inhabit big portions of our genome. Moreover, although they are mainly inert, some of the evolutionarily younger members maintain the ability to express both RNA and proteins. We have developed an approach using long-read single-molecule real-time (SMRT) sequencing that produces long reads that allow us to obtain detailed and accurate HERV-K HML-2 expression profiles. We applied this approach to study HERV-K expression in the presence or absence of productive HIV-1 infection of primary human CD4+ T cells. In addition to using SMRT sequencing, our strategy also includes the magnetic selection of the infected cells so that levels of background expression due to uninfected cells are kept at a minimum. The results presented here provide a blueprint for in-depth studies of the interactions of the authentic upregulated HERV-K HML-2 elements and HIV-1.
Assuntos
Linfócitos T CD4-Positivos/virologia , Retrovirus Endógenos/genética , Regulação Viral da Expressão Gênica , HIV-1/fisiologia , Provírus/genética , Proteínas Virais/genética , Células Cultivadas , Retrovirus Endógenos/fisiologia , Genoma Humano , HIV-1/genética , Humanos , Provírus/fisiologia , RNA Viral/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
BCR-JAK2 is an infrequent gene fusion found in chronic/acute, myeloid/lymphoid Philadelphia chromosome-negative leukaemia. In this study, we demonstrated that in vivo expression of BCR-JAK2 in mice induces neoplasia, with fatal consequences. Transplantation of BCR-JAK2 bone marrow progenitors promoted splenomegaly, with megakaryocyte infiltration and elevated leukocytosis of myeloid origin. Analysis of peripheral blood revealed the presence of immature myeloid cells, platelet aggregates and ineffective erythropoiesis. A possible molecular mechanism for these observations involved inhibition of apoptosis by deregulated expression of the anti-apoptotic mediator Bcl-xL and the serine/threonine kinase Pim1. Together, these data provide a suitable in vivo molecular mechanism for leukaemia induction by BCR-JAK2 that validates the use of this model as a relevant preclinical tool for the design of new targeted therapies in Philadelphia chromosome-negative leukaemia involving BCR-JAK2-driven activation of the JAK2 pathway.
Assuntos
Janus Quinase 2/fisiologia , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Proteínas Proto-Oncogênicas c-bcr/fisiologia , Animais , Feminino , Rearranjo Gênico , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/fisiologia , Janus Quinase 2/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/mortalidade , Leucocitose/etiologia , Masculino , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-bcr/genética , Retroviridae , Fator de Transcrição STAT5/metabolismo , Esplenomegalia/etiologia , Transdução Genética/métodos , TransgenesRESUMO
Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as beta-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall beta-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IkappaB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in beta-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.
Assuntos
Candida albicans/imunologia , Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas do Tecido Nervoso/metabolismo , beta-Glucanas/metabolismo , Candida albicans/genética , Adesão Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Lectinas Tipo C , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/imunologia , Viabilidade Microbiana , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas do Tecido Nervoso/imunologia , Fagocitose , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Baço/imunologia , beta-Glucanas/imunologiaRESUMO
Hematopoietic stem cells (HSCs) interact dynamically with an intricate network of cells in the bone marrow (BM) microenvironment or niche. These interactions provide instructive cues that influence the production and lineage determination of different types of blood cells and maintenance of HSC quiescence. They also contribute to hematopoietic deregulation and hematological myeloid malignancies. Alterations in the BM niche are commonly observed in myeloid malignancies and contribute to the aberrant function of myelodysplastic and leukemia-initiating stem cells. In this work, we review how different components of the BM niche affect normal hematopoiesis, the molecular signals that govern this interaction, and how genetic changes in stromal cells or alterations in remodeled malignant BM niches contribute to myeloid malignancies. Understanding the intricacies between normal and malignant niches and their modulation may provide insights into developing novel therapeutics for blood disorders.
Assuntos
Neoplasias da Medula Óssea/patologia , Medula Óssea/fisiologia , Microambiente Celular/fisiologia , Quimiocina CXCL12/metabolismo , Células Endoteliais/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Mesenquimais/metabolismo , Nestina/metabolismo , Neuroglia/fisiologia , Neurônios/fisiologia , Osteoblastos/fisiologia , Osteócitos/fisiologia , Receptores para Leptina/metabolismo , Nicho de Células-Tronco/fisiologia , Sistema Nervoso Simpático/fisiologiaRESUMO
Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret this change as a consequence of a cease in cell growth, while the ongoing cell divisions leave the cells as single cells. Late in the infection cycle, cells with low-density cell walls appear, and these cells can be detected on cytograms of light scatter versus, for instance, fluorescence of stained DNA. We describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture. The method can be performed in real time and therefore increases the chance of successful intervention in the fermentation process.
Assuntos
Bacteriófagos/crescimento & desenvolvimento , Citometria de Fluxo/métodos , Lactococcus lactis/virologia , Animais , Leite/microbiologia , Reprodutibilidade dos TestesRESUMO
The human endogenous retroviruses (HERV)-K of the HML-2 group include full-length or near full-length elements encoding functional proteins, and are classified as type-1 or type-2 (type-1 has a deletion in the 5' end of the env gene). Because proteins of different retroviruses can interact, we hypothesized that HERV-K envelope (Env) could influence HIV-1 replication. Here we describe the negative effect of envelope expression of certain type-2 HERV-Ks on HIV-1 production. All HIV-1 and SIV strains tested were susceptible to various degrees to inhibition by the HERV-K108 envelope. We identified four residues within HERV-K108 Env as being critical to inhibit HIV-1 production. No inhibition was observed on EGFP expression, indicating that HERV-K Env does not affect general protein production. These findings demonstrate that envelope proteins from some endogenous retroviruses can limit production of exogenous lentiviruses such as HIV-1. Future studies will elucidate the mechanism mediating HIV-1 inhibition by HERV Envs.
Assuntos
Retrovirus Endógenos/genética , Expressão Gênica , HIV-1/crescimento & desenvolvimento , Proteínas do Envelope Viral/biossíntese , Interferência Viral , Análise Mutacional de DNA , Vírus da Imunodeficiência Símia/crescimento & desenvolvimentoRESUMO
Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.