RESUMEN
Recent advances in single-cell omics have transformed characterisation of cell types in challenging-to-study biological contexts. In contexts with limited single-cell samples, such as the early human embryo inference of transcription factor-gene regulatory network (GRN) interactions is especially difficult. Here, we assessed application of different linear or non-linear GRN predictions to single-cell simulated and human embryo transcriptome datasets. We also compared how expression normalisation impacts on GRN predictions, finding that transcripts per million reads outperformed alternative methods. GRN inferences were more reproducible using a non-linear method based on mutual information (MI) applied to single-cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), compared with alternative network prediction methods tested. MICA captures complex non-monotonic dependencies and feedback loops. Using MICA, we generated the first GRN inferences in early human development. MICA predicted co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2γ in early human embryos. Overall, our comparative analysis of GRN prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets in especially challenging contexts to infer interactions between transcription factor expression and target gene regulation.
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Redes Reguladoras de Genes , Multiómica , Humanos , Redes Reguladoras de Genes/genética , Factores de Transcripción/metabolismo , Transcriptoma/genética , Embrión de MamíferosRESUMEN
Our understanding of the molecular events driving cell specification in early mammalian development relies mainly on mouse studies, and it remains unclear whether these mechanisms are conserved across mammals, including humans. We have shown that the establishment of cell polarity via aPKC is a conserved event in the initiation of the trophectoderm (TE) placental programme in mouse, cow and human embryos. However, the mechanisms transducing cell polarity into cell fate in cow and human embryos are unknown. Here, we have examined the evolutionary conservation of Hippo signalling, which is thought to function downstream of aPKC activity, in four different mammalian species: mouse, rat, cow and human. In all four species, inhibition of the Hippo pathway by targeting LATS kinases is sufficient to drive ectopic TE initiation and downregulation of SOX2. However, the timing and localisation of molecular markers differ across species, with rat embryos more closely recapitulating human and cow developmental dynamics, compared with the mouse. Our comparative embryology approach uncovered intriguing differences as well as similarities in a fundamental developmental process among mammals, reinforcing the importance of cross-species investigations.
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Vía de Señalización Hippo , Transducción de Señal , Bovinos , Humanos , Femenino , Embarazo , Ratones , Ratas , Animales , Transducción de Señal/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Blastocisto/metabolismo , Placenta/metabolismo , Mamíferos/metabolismo , Linaje de la CélulaRESUMEN
Current knowledge of the transcriptional regulation of human pluripotency is incomplete, with lack of interspecies conservation observed. Single-cell transcriptomics analysis of human embryos previously enabled us to identify transcription factors, including the zinc-finger protein KLF17, that are enriched in the human epiblast and naïve human embryonic stem cells (hESCs). Here, we show that KLF17 is expressed coincident with the known pluripotency-associated factors NANOG and SOX2 across human blastocyst development. We investigate the function of KLF17 using primed and naïve hESCs for gain- and loss-of-function analyses. We find that ectopic expression of KLF17 in primed hESCs is sufficient to induce a naïve-like transcriptome and that KLF17 can drive transgene-mediated resetting to naïve pluripotency. This implies a role for KLF17 in establishing naïve pluripotency. However, CRISPR-Cas9-mediated knockout studies reveal that KLF17 is not required for naïve pluripotency acquisition in vitro. Transcriptome analysis of naïve hESCs identifies subtle effects on metabolism and signalling pathways following KLF17 loss of function, and possible redundancy with other KLF paralogues. Overall, we show that KLF17 is sufficient, but not necessary, for naïve pluripotency under the given in vitro conditions.
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Blastocisto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Factores de Transcripción/metabolismo , Humanos , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genéticaRESUMEN
CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.
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Sistemas CRISPR-Cas , Células Madre Pluripotentes , Sistemas CRISPR-Cas/genética , Edición Génica , Humanos , Mutación con Pérdida de Función , ARN Guía de Kinetoplastida/genéticaRESUMEN
CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in â¼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.
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Blastocisto/metabolismo , Sistemas CRISPR-Cas , Edición Génica , Células Madre Embrionarias Humanas/metabolismo , Pérdida de Heterocigocidad , Factor 3 de Transcripción de Unión a Octámeros , Línea Celular , Cromosomas Humanos Par 6/genética , Cromosomas Humanos Par 6/metabolismo , HumanosRESUMEN
Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.
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Cromosomas de los Mamíferos/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Pluripotentes/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/deficiencia , Animales , Blastocisto/citología , Blastocisto/metabolismo , Supervivencia Celular , Cromosomas de los Mamíferos/genética , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.
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Evolución Biológica , Ectodermo/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Transcripción Genética , Trofoblastos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Masa Celular Interna del Blastocisto/citología , Masa Celular Interna del Blastocisto/metabolismo , Bovinos , Linaje de la Célula , Polaridad Celular , Ectodermo/citología , Embrión de Mamíferos/enzimología , Femenino , Factor de Transcripción GATA3/metabolismo , Vía de Señalización Hippo , Humanos , Ratones , Mórula/citología , Mórula/enzimología , Mórula/metabolismo , Placenta/citología , Placenta/metabolismo , Embarazo , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Trofoblastos/citología , Proteínas Señalizadoras YAP , Saco Vitelino/citología , Saco Vitelino/metabolismoRESUMEN
DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.
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Mutación , Oocitos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reparación del ADN por Recombinación/genética , Aneuploidia , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Femenino , Fertilización , Genes bcl-2 , Meiosis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/citología , Proteínas de Unión a Fosfato/deficiencia , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.
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Autorrenovación de las Células/fisiología , Células Madre Embrionarias Humanas/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Activinas/metabolismo , Animales , Blastocisto/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Medios de Cultivo/farmacología , Endodermo/citología , Endodermo/metabolismo , Membranas Extraembrionarias/citología , Membranas Extraembrionarias/metabolismo , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Transcriptoma , Inactivación del Cromosoma X/fisiologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0004264.].
RESUMEN
This corrects the article DOI: 10.1038/nature24033.
RESUMEN
Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.
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Desarrollo Embrionario/genética , Edición Génica , Regulación del Desarrollo de la Expresión Génica , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Animales , Blastocisto/metabolismo , Sistemas CRISPR-Cas/genética , Linaje de la Célula , Ectodermo/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Estratos Germinativos/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Masculino , Ratones , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/deficiencia , Especificidad por Sustrato , Cigoto/metabolismoRESUMEN
Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.
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Carcinogénesis , Proliferación Celular , Quinasas Asociadas a rho/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Técnicas de Inactivación de Genes , Melanoma/patología , Ratones , Quinasas Asociadas a rho/genéticaRESUMEN
Polarization of cells into a protrusive front and a retracting cell body is the hallmark of mesenchymal-like cell migration. Many mRNAs are localized to protrusions, but it is unclear to what degree mRNA localization contributes toward protrusion formation. We performed global quantitative analysis of the distributions of mRNAs, proteins, and translation rates between protrusions and the cell body by RNA sequencing (RNA-seq) and quantitative proteomics. Our results reveal local translation as a key determinant of protein localization to protrusions. Accordingly, inhibition of local translation destabilizes protrusions and inhibits mesenchymal-like morphology. Interestingly, many mRNAs localized to protrusions are translationally repressed. Specific cis-regulatory elements within mRNA UTRs define whether mRNAs are locally translated or repressed. Finally, RNAi screening of RNA-binding proteins (RBPs) enriched in protrusions revealed trans-regulators of localized translation that are functionally important for protrusions. We propose that by deciphering the localized mRNA UTR code, these proteins regulate protrusion stability and mesenchymal-like morphology.
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Movimiento Celular/genética , Extensiones de la Superficie Celular/metabolismo , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Unión Proteica/genética , Transporte de Proteínas , Proteínas de Unión al ARN/genética , Análisis de Secuencia de ARN/métodosRESUMEN
There is an urgent need to identify new therapeutic opportunities for metastatic melanoma. Fragment-based screening has led to the discovery of orally available, ATP-competitive AKT kinase inhibitors, AT13148 and CCT129254. These compounds also inhibit the Rho-kinases ROCK 1 and ROCK 2 and we show they potently inhibit ROCK activity in melanoma cells in culture and in vivo. Treatment of melanoma cells with CCT129254 or AT13148 dramatically reduces cell invasion, impairing both "amoeboid-like" and mesenchymal-like modes of invasion in culture. Intravital imaging shows that CCT129254 or AT13148 treatment reduces the motility of melanoma cells in vivo. CCT129254 inhibits melanoma metastasis when administered 2 days after orthotopic intradermal injection of the cells, or when treatment starts after metastases have arisen. Mechanistically, our data suggest that inhibition of ROCK reduces the ability of melanoma cells to efficiently colonize the lungs. These results suggest that these novel inhibitors of ROCK may be beneficial in the treatment of metastasis.
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2-Hidroxifenetilamina/análogos & derivados , Movimiento Celular/efectos de los fármacos , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirazoles/administración & dosificación , Quinasas Asociadas a rho/genética , 2-Hidroxifenetilamina/administración & dosificación , Línea Celular Tumoral , Humanos , Melanoma/genética , Melanoma/patología , Invasividad Neoplásica/genética , Metástasis de la Neoplasia , Fosforilación , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell-cell junctions, luminal infiltration and basal invasion induced by AGE-modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180(Δ) (Ex2-6/) (Δ) (Ex2-6) mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Membrana Basal/metabolismo , Movimiento Celular , Células Epiteliales/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Lectinas de Unión a Manosa/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Mitogénicos/metabolismo , Animales , Membrana Basal/patología , Línea Celular Tumoral , Supervivencia Celular , Elasticidad , Humanos , Estimación de Kaplan-Meier , Lectinas Tipo C/metabolismo , Masculino , Mecanotransducción Celular , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones Noqueados , Invasividad Neoplásica , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/patología , Estructura Terciaria de Proteína , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Factores de TiempoRESUMEN
Phospholipase Cε (PLCε) has been characterized as a direct effector of Ras in vitro and in cellular systems; however, the role of PLCε in tumorigenesis and its link to Ras in this context remain unclear. To assess the role of PLCε in Ras-driven cancers, we generated two new mouse strains: one carrying a targeted deletion of Plce (Plce(-/-)) and the other carrying mutant alleles of Plce unable to bind to Ras (Plce(RAm/RAm)). The Plce(-/-) and, to a lesser degree, Plce(RAm/RAm) transgenic mice exhibited increased susceptibility to tumor formation in the two-stage skin carcinogenesis protocol, revealing a tumor suppressor function for this PLC. This result also suggests that in this context Ras binding in part regulates functions of PLCε. Although significant differences were not seen in the LSL-Kras(G12D) nonsmall cell lung carcinoma model, down-regulation of PLCε was found in animal tumors and in cellular systems following expression of the oncogenic Ras. An inhibitory impact of PLCε on cell growth requires intact lipase activity and is likely mediated by protein kinase C enzymes. Further cellular studies suggest involvement of histone deacetylase in the mechanism of PLCε down-regulation. Taken together, our results show a previously unidentified tumor suppressor role for this PLC in animal models and, together with observations of marked down-regulation in colorectal, lung, and skin tumors, suggest its use as a biological marker in cancer.
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Regulación Neoplásica de la Expresión Génica/fisiología , Genes Supresores de Tumor/fisiología , Genes ras/genética , Neoplasias/genética , Fosfoinositido Fosfolipasa C/fisiología , Animales , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos , Fosfoinositido Fosfolipasa C/genética , Proteína Quinasa C/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Cell fate determination in the progeny of mammary epithelial stem/progenitor cells remains poorly understood. Here, we have examined the role of the mitotic kinase Aurora A (AURKA) in regulating the balance between basal and luminal mammary lineages. We find that AURKA is highly expressed in basal stem cells and, to a lesser extent, in luminal progenitors. Wild-type AURKA expression promoted luminal cell fate, but expression of an S155R mutant reduced proliferation, promoted basal fate, and inhibited serial transplantation. The mechanism involved regulation of mitotic spindle orientation by AURKA and the positioning of daughter cells after division. Remarkably, this was NOTCH dependent, as NOTCH inhibitor blocked the effect of wild-type AURKA expression on spindle orientation and instead mimicked the effect of the S155R mutant. These findings directly link AURKA, NOTCH signaling, and mitotic spindle orientation and suggest a mechanism for regulating the balance between luminal and basal lineages in the mammary gland.
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Aurora Quinasa A/metabolismo , Linaje de la Célula , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Receptor Notch1/metabolismo , Huso Acromático/metabolismo , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Aurora Quinasa A/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Proliferación Celular , Células Epiteliales/citología , Femenino , Glándulas Mamarias Animales/citología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Unión ProteicaRESUMEN
The way in which cells adopt different morphologies is not fully understood. Cell shape could be a continuous variable or restricted to a set of discrete forms. We developed quantitative methods to describe cell shape and show that Drosophila haemocytes in culture are a heterogeneous mixture of five discrete morphologies. In an RNAi screen of genes affecting the morphological complexity of heterogeneous cell populations, we found that most genes regulate the transition between discrete shapes rather than generating new morphologies. In particular, we identified a subset of genes, including the tumour suppressor PTEN, that decrease the heterogeneity of the population, leading to populations enriched in rounded or elongated forms. We show that these genes have a highly conserved function as regulators of cell shape in both mouse and human metastatic melanoma cells.
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Forma de la Célula , Drosophila melanogaster/metabolismo , Genes Supresores de Tumor , Pruebas Genéticas , Melanoma/secundario , Interferencia de ARN , Animales , Drosophila melanogaster/genética , Humanos , Melanoma/genética , Melanoma/metabolismo , Ratones , Fosfohidrolasa PTEN/genética , Fenotipo , Células Tumorales CultivadasRESUMEN
RAL small GTPases, encoded by the Rala and Ralb genes, are members of the RAS superfamily of small GTPases and can act as downstream effectors of RAS [1]. Although highly similar, distinct functions have been identified for RALA and RALB: RALA has been implicated in epithelial cell polarity [2], insulin secretion [3], GLUT4 translocation [4, 5], neurite branching, and neuronal polarity [6, 7], and RALB in tumor cell survival [8], migration/invasion [9-12], TBK1 activation [13], and autophagy [14]. To investigate RAL GTPases in vivo, we generated null and conditional knockout mice. Ralb null mice are viable with no overt phenotype; the Rala null leads to exencephaly and embryonic lethality. The exencephaly phenotype is exacerbated in Rala(-/-);Ralb(+/-) embryos; embryos null for Rala and Ralb do not live past gastrulation. Using a Kras-driven non-small cell lung carcinoma mouse model, we found that either RALA or RALB is sufficient for tumor growth. However, deletion of both Ral genes blocks tumor formation. Either RALA or RALB is sufficient for cell proliferation, but cells lacking both fail to proliferate. These studies demonstrate functions of RAL proteins in development, tumorigenesis, and cell proliferation and show that RALA and RALB act in a redundant fashion.