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1.
PLoS Biol ; 20(3): e3001596, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35353806

RESUMEN

Hedgehog (HH) signaling is important for embryonic pattering and stem cell differentiation. The G protein-coupled receptor (GPCR) Smoothened (SMO) is the key HH signal transducer modulating both transcription-dependent and transcription-independent responses. We show that SMO protects naive mouse embryonic stem cells (ESCs) from dissociation-induced cell death. We exploited this SMO dependency to perform a genetic screen in haploid ESCs where we identify the Golgi proteins TMED2 and TMED10 as factors for SMO regulation. Super-resolution microscopy shows that SMO is normally retained in the endoplasmic reticulum (ER) and Golgi compartments, and we demonstrate that TMED2 binds to SMO, preventing localization to the plasma membrane. Mutation of TMED2 allows SMO accumulation at the plasma membrane, recapitulating early events after HH stimulation. We demonstrate the physiologic relevance of this interaction in neural differentiation, where TMED2 functions to repress HH signal strength. Identification of TMED2 as a binder and upstream regulator of SMO opens the way for unraveling the events in the ER-Golgi leading to HH signaling activation.


Asunto(s)
Proteínas Hedgehog , Receptores Acoplados a Proteínas G , Animales , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de la Membrana , Ratones , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas de Transporte Vesicular
2.
Mol Cell ; 56(5): 617-29, 2014 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-25454946

RESUMEN

Inflammation is a significant factor in cancer development, and a molecular understanding of the parameters dictating the impact of inflammation on cancers could significantly improve treatment. The tumor suppressor p53 is frequently mutated in cancer, and p53 missense mutants (mutp53) can acquire oncogenic properties. We report that cancer cells with mutp53 respond to inflammatory cytokines increasing their invasive behavior. Notably, this action is coupled to expression of chemokines that can expose the tumor to host immunity, potentially affecting response to therapy. Mechanistically, mutp53 fuels NF-κB activation while it dampens activation of ASK1/JNK by TNFα, and this action depends on mutp53 binding and inhibiting the tumor suppressor DAB2IP in the cytoplasm. Interfering with such interaction reduced aggressiveness of cancer cells in xenografts. This interaction is an unexplored mechanism by which mutant p53 can influence tumor evolution, with implications for our understanding of the complex role of inflammation in cancer.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Femenino , Células HCT116 , Humanos , Metástasis Linfática , Neoplasias Mamarias Experimentales , Ratones , Ratones SCID , Mutación Missense
3.
Proc Natl Acad Sci U S A ; 114(29): 7623-7628, 2017 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-28667123

RESUMEN

Obesity and type 2 diabetes are significant risk factors for malignancies, being associated with chronic inflammation and hyperinsulinemia. In this context, insulin can synergize with inflammation to promote proliferation, survival, and dissemination of cancer cells. Point mutation of p53 is a frequent event and a significant factor in cancer development and progression. Mutant p53 protein(s) (mutp53) can acquire oncogenic properties that increase metastasis, proliferation, and cell survival. We report that breast and prostate cancer cells with mutant p53 respond to insulin stimulation by increasing cell proliferation and invasivity, and that such a response depends on the presence of mutp53. Mechanistically, we find that mutp53 augments insulin-induced AKT1 activation by binding and inhibiting the tumor suppressor DAB2IP (DAB2-interacting protein) in the cytoplasm. This molecular axis reveals a specific gain of function for mutant p53 in the response to insulin stimulation, offering an additional perspective to understand the relationship between hyperinsulinemia and cancer evolution.


Asunto(s)
Insulina/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-akt/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Complicaciones de la Diabetes/tratamiento farmacológico , Diabetes Mellitus/tratamiento farmacológico , Progresión de la Enfermedad , Femenino , Humanos , Hiperinsulinismo/metabolismo , Inflamación , Masculino , Ratones , Proteínas Mutantes/genética , Obesidad/complicaciones , Obesidad/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Riesgo , Proteínas Activadoras de ras GTPasa/antagonistas & inhibidores
4.
PLoS Comput Biol ; 14(1): e1005950, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29337991

RESUMEN

Haploid cells are increasingly used for screening of complex pathways in animal genomes. Hemizygous mutations introduced through viral insertional mutagenesis can be directly selected for phenotypic changes. Here we present HaSAPPy a tool for analysing sequencing datasets of screens using insertional mutations in large pools of haploid cells. Candidate gene prediction is implemented through identification of enrichment of insertional mutations after selection by simultaneously evaluating several parameters. We have developed HaSAPPy for analysis of genetic screens for silencing factors of X chromosome inactivation in haploid mouse embryonic stem cells. To benchmark the performance, we further analyse several datasets of genetic screens in human haploid cells for which candidates have been validated. Our results support the effective candidate prediction strategy of HaSAPPy. HaSAPPy is implemented in Python, licensed under the MIT license, and is available from https://github.com/gdiminin/HaSAPPy.


Asunto(s)
Biología Computacional/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Algoritmos , Alelos , Animales , Gráficos por Computador , Biblioteca de Genes , Genoma Humano , Haploidia , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Mutagénesis Insercional , Mutación , Fenotipo , Picornaviridae/genética , Lenguajes de Programación
5.
PLoS One ; 19(6): e0301670, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38917070

RESUMEN

The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we show that Spinal Cord Organoids (SCOs) can be applied to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. We validate this system by measuring the effects of mutating the HH receptor PTCH1 and the impact of HH agonists and antagonists on NPC specification. SCOs represent an accessible and reliable in-vitro tool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation.


Asunto(s)
Proteínas Hedgehog , Organoides , Transducción de Señal , Médula Espinal , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Animales , Organoides/metabolismo , Organoides/citología , Médula Espinal/metabolismo , Médula Espinal/citología , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Receptor Patched-1/metabolismo , Receptor Patched-1/genética
6.
Proc Natl Acad Sci U S A ; 107(14): 6322-7, 2010 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-20308539

RESUMEN

The genome of the fruitfly Drosophila melanogaster contains a single p53-like protein, phylogenetically related to the ancestor of the mammalian p53 family of tumor suppressors. We reasoned that a comprehensive map of the protein interaction profile of Drosophila p53 (Dmp53) might help identify conserved interactions of the entire p53 family in man. Using a genome-scale in vitro expression cloning approach, we identified 91 previously unreported Dmp53 interactors, considerably expanding the current Drosophila p53 interactome. Looking for evolutionary conservation of these interactions, we tested 41 mammalian orthologs and found that 37 bound to one or more p53-family members when overexpressed in human cells. An RNAi-based functional assay for modulation of the p53 pathway returned five positive hits, validating the biological relevance of these interactions. One p53 interactor is GTPBP4, a nucleolar protein involved in 60S ribosome biogenesis. We demonstrate that GTPBP4 knockdown induces p53 accumulation and activation in the absence of nucleolar disruption. In breast tumors with wild-type p53, increased expression of GTPBP4 correlates with reduced patient survival, emphasizing a potential relevance of this regulatory axis in cancer.


Asunto(s)
Clonación Molecular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Redes Reguladoras de Genes , Genoma , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Evolución Molecular , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteína p53 Supresora de Tumor/genética
7.
Stem Cell Reports ; 17(1): 43-52, 2022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-34919812

RESUMEN

Mammalian haploid cells have applications for genetic screening and substituting gametic genomes. Here, we characterize a culture system for obtaining haploid primordial germ cell-like cells (PGCLCs) from haploid mouse embryonic stem cells (ESCs). We find that haploid cells show predisposition for PGCLCs, whereas a large fraction of somatic cells becomes diploid. Characterization of the differentiating haploid ESCs (haESCs) reveals that Xist is activated from and colocalizes with the single X chromosome. This observation suggests that X chromosome inactivation (XCI) is initiated in haploid cells consistent with a model where autosomal blocking factors set a threshold for X-linked activators. We further find that Xist expression is lost at later timepoints in differentiation, which likely reflects the loss of X-linked activators. In vitro differentiation of haploid PGCLCs can be a useful approach for future studies of potential X-linked activators of Xist.


Asunto(s)
Células Madre Embrionarias/metabolismo , Células Germinativas/metabolismo , Haploidia , ARN Largo no Codificante/genética , Cromosoma X , Animales , Biomarcadores , Diferenciación Celular/genética , Células Madre Embrionarias/citología , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Ratones , Inactivación del Cromosoma X
8.
PLoS One ; 15(9): e0233072, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32911495

RESUMEN

In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of adult mammal development by natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for mammalian development. Haploid embryonic stem cells (haESCs) can be established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitutes of gametic genomes, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletion of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further, we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs yield semi-cloned embryos and mice when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improving the development of semi-cloned embryos derived by haESC injection.


Asunto(s)
Clonación de Organismos/métodos , Desarrollo Embrionario , Haploidia , Partenogénesis , Poliploidía , Animales , Línea Celular , Células Madre Embrionarias/citología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA
9.
Biochem Biophys Res Commun ; 388(2): 428-33, 2009 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-19666006

RESUMEN

C16orf35 is a highly conserved gene positioned upstream of the alpha-globins in humans and other vertebrates. The deduced protein is also highly conserved, it has no defined structural features or domains, and its function is currently unknown. Here we show that the C16orf35 protein has nuclear and cytosolic distribution, and can localize to PML nuclear bodies. The C16orf35 protein was detected in several human transformed cells lines, and studies of transient and stable overexpression indicate that increased levels of C16orf35 inhibit cell proliferation. We also find that C16orf35 interacts with human p73, and represses transcription by TAp73gamma but not by TAp73alpha. This selectivity is not due to differential interaction, since C16orf35 binds both p73 variants. Our data suggest that C16orf35 can modulate differentially the specific activities of selected p73 isoforms.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular , Secuencia Conservada , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Evolución Molecular , Proteínas Activadoras de GTPasa , Humanos , Proteínas Nucleares/genética , Activación Transcripcional , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genética
10.
Sci Adv ; 5(9): eaaw6490, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31555730

RESUMEN

Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4-Å cryo-EM structure of the human PTCH1 bound to ShhNC24II, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by 10 sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol-sensing domain (SSD) and the SSD-like domain (SSDL), which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.


Asunto(s)
Proteínas Hedgehog/química , Membrana Dobles de Lípidos/química , Receptor Patched-1/química , Esteroles/química , Transporte Biológico , Microscopía por Crioelectrón , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/ultraestructura , Humanos , Membrana Dobles de Lípidos/metabolismo , Receptor Patched-1/metabolismo , Receptor Patched-1/ultraestructura , Dominios Proteicos , Esteroles/metabolismo
11.
Methods Mol Biol ; 1861: 1-18, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30218355

RESUMEN

Forward genetics can provide insight into molecular pathways as has been demonstrated by advances in cell biology from comprehensive genetic studies in simple organisms. Recently, techniques have become available that promise a similar potential for understanding developmental pathways in mammals. Here we describe a genetic forward screening approach for identifying factors involved in X chromosome inactivation that is based on haploid mouse embryonic stem cells. Using a genetically encoded selection system screening of large mutant pools can identify candidate silencing factors with high confidence.


Asunto(s)
Técnicas Genéticas , Células Madre Embrionarias de Ratones , Inactivación del Cromosoma X , Animales , Línea Celular , Epigenómica/métodos , Femenino , Células HEK293 , Haploidia , Humanos , Ratones
12.
Cell Death Differ ; 25(7): 1224-1238, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29568059

RESUMEN

The tumor suppressor DAB2IP contributes to modulate the network of information established between cancer cells and tumor microenvironment. Epigenetic and post-transcriptional inactivation of this protein is commonly observed in multiple human malignancies, and can potentially favor progression of tumors driven by a variety of genetic mutations. Performing a high-throughput screening of a large collection of human microRNA mimics, we identified miR-149-3p as a negative post-transcriptional modulator of DAB2IP. By efficiently downregulating DAB2IP, this miRNA enhances cancer cell motility and invasiveness, facilitating activation of NF-kB signaling and promoting expression of pro-inflammatory and pro-angiogenic factors. In addition, we found that miR-149-3p secreted by prostate cancer cells induces DAB2IP downregulation in recipient vascular endothelial cells, stimulating their proliferation and motility, thus potentially remodeling the tumor microenvironment. Finally, we found that inhibition of endogenous miR-149-3p restores DAB2IP activity and efficiently reduces tumor growth and dissemination of malignant cells. These observations suggest that miR-149-3p can promote cancer progression via coordinated inhibition of DAB2IP in tumor cells and in stromal cells.


Asunto(s)
MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/metabolismo , ARN Neoplásico/metabolismo , Transducción de Señal , Microambiente Tumoral , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Células HCT116 , Células HeLa , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , MicroARNs/genética , Proteínas de Neoplasias/genética , Células PC-3 , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Neoplásico/genética , Pez Cebra , Proteínas Activadoras de ras GTPasa/genética
13.
Cell Death Differ ; 24(1): 15-25, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27858941

RESUMEN

One of the most defining features of cancer is aberrant cell communication; therefore, a molecular understanding of the intricate network established among tumor cells and their microenvironment could significantly improve comprehension and clinical management of cancer. The tumor suppressor DAB2IP (Disabled homolog 2 interacting protein), also known as AIP1 (ASK1 interacting protein), has an important role in this context, as it modulates signal transduction by multiple inflammatory cytokines and growth factors. DAB2IP is a Ras-GAP, and negatively controls Ras-dependent mitogenic signals. In addition, acting as a signaling adaptor, DAB2IP modulates other key oncogenic pathways, including TNFα/NF-κB, WNT/ß-catenin, PI3K/AKT, and androgen receptors. Therefore, DAB2IP inactivation can provide a selective advantage to tumors initiated by a variety of driver mutations. In line with this role, DAB2IP expression is frequently impaired by methylation in cancer. Interestingly, recent studies reveal that tumor cells can employ other sophisticated mechanisms to disable DAB2IP at the post-transcriptional level. We review the mechanisms and consequences of DAB2IP inactivation in cancer, with the purpose to support and improve research aimed to counteract such mechanisms. We suggest that DAB2IP reactivation in cancer cells could be a strategy to coordinately dampen multiple oncogenic pathways, potentially limiting progression of a wide spectrum of tumors.


Asunto(s)
Neoplasias/patología , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Resistencia a Antineoplásicos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/genética
14.
Mol Cell Oncol ; 2(4): e1002719, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-27308497

RESUMEN

Inflammation and mutation of the tumor suppressor p53 are two apparently unrelated conditions that are strongly associated with cancer initiation and progression. We recently reported that gain-of-function mutant p53 modifies the response of cancer cells to inflammatory signals by binding a cytoplasmic tumor suppressor protein involved in TNFα signaling.

15.
Cell Rep ; 12(4): 554-61, 2015 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-26190100

RESUMEN

In mammals, the noncoding Xist RNA triggers transcriptional silencing of one of the two X chromosomes in female cells. Here, we report a genetic screen for silencing factors in X chromosome inactivation using haploid mouse embryonic stem cells (ESCs) that carry an engineered selectable reporter system. This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist. Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends. Independent validation through gene deletion in ESCs confirms that Spen is required for gene repression by Xist. However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2. The identification of Spen opens avenues for further investigation into the gene-silencing pathway of Xist and shows the usefulness of haploid ESCs for genetic screening of epigenetic pathways.


Asunto(s)
Células Madre Embrionarias/metabolismo , Silenciador del Gen , Proteínas Nucleares/metabolismo , ARN Largo no Codificante/genética , Animales , Células Cultivadas , Proteínas de Unión al ADN , Proteína Potenciadora del Homólogo Zeste 2 , Haploidia , Ratones , Proteínas Nucleares/genética , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas de Unión al ARN
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