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1.
Mod Pathol ; 35(11): 1702-1712, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35798968

RESUMEN

Endometrial polyps (EMPs) are common exophytic masses associated with abnormal uterine bleeding and infertility. Unlike normal endometrium, which is cyclically shed, EMPs persist over ovulatory cycles and after the menopause. Despite their usual classification as benign entities, EMPs are paradoxically associated with endometrial carcinomas of diverse histologic subtypes, which frequently arise within EMPs. The etiology and potential origins of EMPs as clonally-derived neoplasms are uncertain, but previous investigations suggested that EMPs are neoplasms of stromal origin driven by recurring chromosomal rearrangements. To better define benign EMPs at the molecular genetic level, we analyzed individual EMPs from 31 women who underwent hysterectomy for benign indications. The 31 EMPs were subjected to comprehensive genomic profiling by exome sequencing of a large panel of tumor-related genes including oncogenes, tumor suppressors, and chromosomal translocation partners. There were no recurring chromosomal rearrangements, and copy-number analyses did not reveal evidence of significant chromosome-level events. Surprisingly, there was a high incidence of single nucleotide variants corresponding to classic oncogenic drivers (i.e., definitive cancer drivers). The spectrum of known oncogenic driver events matched that of endometrial cancers more closely than any other common cancer. Further analyses including laser-capture microdissection showed that these mutations were present in the epithelial compartment at low allelic frequencies. These results establish a link between EMPs and the acquisition of endometrial cancer driver mutations. Based on these findings, we propose a model where the association between EMPs and endometrial cancer is explained by the age-related accumulation of endometrial cancer drivers in a protected environment that-unlike normal endometrium-is not subject to cyclical shedding.


Asunto(s)
Neoplasias Endometriales , Pólipos , Neoplasias Uterinas , Femenino , Humanos , Recurrencia Local de Neoplasia/patología , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Pólipos/genética , Pólipos/patología , Neoplasias Uterinas/patología , Mutación , Carcinogénesis/patología , Nucleótidos , Endometrio/patología
2.
J Pathol ; 254(1): 20-30, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33506979

RESUMEN

The endometrium is unique as an accessible anatomic location that can be repeatedly biopsied and where diagnostic biopsies do not extirpate neoplastic lesions. We exploited these features to retrospectively characterize serial genomic alterations along the precancer/cancer continuum in individual women. Cases were selected based on (1) endometrial cancer diagnosis/hysterectomy and (2) preceding serial endometrial biopsies including for some patients an early biopsy before a precancer histologic diagnosis. A comprehensive panel was designed for endometrial cancer genes. Formalin-fixed, paraffin-embedded specimens for each cancer, preceding biopsies, and matched germline samples were subjected to barcoded high-throughput sequencing to identify mutations and track their origin and allelic frequency progression. In total, 92 samples from 21 patients were analyzed, providing an opportunity for new insights into early endometrial cancer progression. Definitive invasive endometrial cancers exhibited expected mutational spectra, and canonical driver mutations were detectable in preceding biopsies. Notably, ≥1 cancer mutations were detected prior to the histopathologic diagnosis of an endometrial precancer in the majority of patients. In 18/21 cases, ≥1 mutations were confirmed by abnormal protein levels or subcellular localization by immunohistochemistry, confirming genomic data and providing unique views of histologic correlates. In 19 control endometria, mutation counts were lower, with a lack of canonical endometrial cancer hotspot mutations. Our study documents the existence of endometrial lesions that are histologically indistinct but are bona fide endometrial cancer precursors. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.


Asunto(s)
Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Endometrio/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Adulto , Anciano , Análisis Mutacional de ADN/métodos , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Análisis de Secuencia de ADN/métodos
3.
Proc Natl Acad Sci U S A ; 116(51): 25880-25890, 2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31772025

RESUMEN

Uterine carcinosarcoma is an aggressive variant of endometrial carcinoma characterized by unusual histologic features including discrete malignant epithelial and mesenchymal components (carcinoma and sarcoma). Recent studies have confirmed a monoclonal origin, and comprehensive genomic characterizations have identified mutations such as Tp53 and Pten However, the biological origins and specific combination of driver events underpinning uterine carcinosarcoma have remained mysterious. Here, we explored the role of the tumor suppressor Fbxw7 in endometrial cancer through defined genetic model systems. Inactivation of Fbxw7 and Pten resulted in the formation of precancerous lesions (endometrioid intraepithelial neoplasia) and well-differentiated endometrioid adenocarcinomas. Surprisingly, all adenocarcinomas eventually developed into definitive uterine carcinosarcomas with carcinomatous and sarcomatous elements including heterologous differentiation, yielding a faithful genetically engineered model of this cancer type. Genomic analysis showed that most tumors spontaneously acquired Trp53 mutations, pointing to a triad of pathways (p53, PI3K, and Fbxw7) as the critical combination underpinning uterine carcinosarcoma, and to Fbxw7 as a key driver of this enigmatic endometrial cancer type. Lineage tracing provided formal genetic proof that the uterine carcinosarcoma cell of origin is an endometrial epithelial cell that subsequently undergoes a prominent epithelial-mesenchymal transition underlying the attainment of a highly invasive phenotype specifically driven by Fbxw7.


Asunto(s)
Neoplasias Endometriales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Neoplasias Uterinas/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Endometrio/patología , Células Epiteliales , Femenino , Humanos , Ratones , Mutación , Fosfohidrolasa PTEN/metabolismo , Fenotipo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/patología
4.
Int J Gynecol Pathol ; 38(6): 503-513, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-30256235

RESUMEN

The diagnosis of endometrioid intraepithelial neoplasia (EIN) is challenging owing to limited sampling, hormonal status, and other confounding histologic variables. Markers such as PTEN or PAX2 can delineate EIN in some cases, but are not wholly reliable. Clearly, new markers of EIN are needed. We explored several potential markers of EIN based rationally on molecular pathways most frequently misregulated in endometrial cancer: the 3-phosphoinositide kinase (PI3K)/AKT, ß-catenin, and mismatch repair pathways. We studied PTEN, PAX2, ß-catenin, and MLH1, in conjunction with 2 new markers-FOXO1 and phosphorylated AKT (pAKT)-not previously investigated in EIN. Benign (n=14) and EIN (n=35) endometria were analyzed by immunohistochemistry. Staining patterns were interpreted, tabulated, and scored by "clonal distinctiveness" in neoplastic lesions; that is, pattern alterations relative to normal glands. In normal endometria, FOXO1 was cytoplasmic in proliferative phase, but nuclear in secretory phase, showing that PI3K/FOXO1 participates in endometrial cycling and that FOXO1 is a readout of PI3K status. pAKT expression was low across normal endometria. FOXO1 or pAKT expression was altered in the majority of EINs (27/35, 77%), with FOXO1 and pAKT being co-altered only in some (20/35, 57%). ß-catenin or MLH1 also exhibited clonal distinctiveness in EINs, showing that these are also useful markers in some cases. This is the first study to demonstrate the potential of pAKT and FOXO1 as biomarkers in the histopathologic evaluation of EIN. However, variability in expression poses challenges in interpretation.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma in Situ/metabolismo , Hiperplasia Endometrial/metabolismo , Neoplasias Endometriales/metabolismo , Proteína Forkhead Box O1/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Carcinoma in Situ/patología , Hiperplasia Endometrial/patología , Neoplasias Endometriales/patología , Endometrio/metabolismo , Endometrio/patología , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Factor de Transcripción PAX2/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Transducción de Señal , beta Catenina/metabolismo
5.
PLoS Genet ; 12(8): e1006215, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27500836

RESUMEN

In mammals, females are born with finite numbers of oocytes stockpiled as primordial follicles. Oocytes are "reawakened" via an ovarian-intrinsic process that initiates their growth. The forkhead transcription factor Foxo3 controls reawakening downstream of PI3K-AKT signaling. However, the identity of the presumptive upstream cell surface receptor controlling the PI3K-AKT-Foxo3 axis has been questioned. Here we show that the receptor tyrosine kinase Kit controls reawakening. Oocyte-specific expression of a novel constitutively-active KitD818V allele resulted in female sterility and ovarian failure due to global oocyte reawakening. To confirm this result, we engineered a novel loss-of-function allele, KitL. Kit inactivation within oocytes also led to premature ovarian failure, albeit via a contrasting phenotype. Despite normal initial complements of primordial follicles, oocytes remained dormant with arrested oocyte maturation. Foxo3 protein localization in the nucleus versus cytoplasm explained both mutant phenotypes. These genetic studies provide formal genetic proof that Kit controls oocyte reawakening, focusing future investigations into the causes of primary ovarian insufficiency and ovarian aging.


Asunto(s)
Proteína Forkhead Box O3/genética , Fosfatidilinositol 3-Quinasas/genética , Insuficiencia Ovárica Primaria/genética , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Núcleo Celular/genética , Citoplasma/genética , Femenino , Proteína Forkhead Box O3/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Insuficiencia Ovárica Primaria/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo
6.
J Assist Reprod Genet ; 32(12): 1741-7, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26507072

RESUMEN

PURPOSE: Foxo3 protein is required in the oocyte nucleus for the maintenance of primordial follicles in a dormant state. PI3K/AKT-dependent phosphorylation of Foxo3 leads to its relocalization to the cytoplasm and subsequent follicular activation. However, the nature of the upstream signals controlling Foxo3 activity and subcellular localization remains unknown. We aimed to study the in vitro effects of Kit ligand (stem cell factor) on the subcellular localization of Foxo3 in primordial follicles within the postnatal mouse ovary. METHODS: This was an in vitro study using explants of intact neonatal mouse ovaries. The study was performed in laboratory animal facility and basic science research laboratory at a University Hospital. The animals used for this study were FVB mice. Neonatal FVB mice ovaries at postnatal day 7 (PD7) were harvested and incubated in culture medium (DMEM) at 37 °C and 5 % CO(2) for 60-90 min with (n = 3) or without (n = 3) Kit ligand at 150 ng/mL (8 nM). Similar experimental conditions were used to establish a dose-response curve for the effects of Kit ligand and assess the effects of imatinib (small molecule inhibitor of the Kit receptor). Immunofluorescence was used to identify the subcellular location of Foxo3 in oocytes. Proportions of cytoplasmic versus nuclear Foxo3 in primordial follicles were determined. RESULTS: Kit ligand treatment increased the cytoplasmic localization of Foxo3 from 40 % in the untreated ovaries to 74 % in the treated group (p = 0.007 in paired samples and p = 0.03 in unpaired samples). Furthermore, this effect was reversible with imatinib (p = 0.005). A dose-response curve for Kit ligand treatment showed that maximum effect was seen at 150 ng/mL. CONCLUSION: Kit ligand treatment in vitro increases the proportion of cytoplasmic Foxo3 in primordial follicles at PD7, lending support to the idea that Kit receptor/ligand controls Foxo3 activity in the context of primordial follicle activation.


Asunto(s)
Factores de Transcripción Forkhead/fisiología , Ovario/metabolismo , Factor de Células Madre/fisiología , Animales , Femenino , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/análisis , Factores de Transcripción Forkhead/metabolismo , Mesilato de Imatinib/farmacología , Técnicas In Vitro , Ratones , Oocitos/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Factor de Células Madre/metabolismo
7.
Cancer Cell ; 41(10): 1731-1748.e8, 2023 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-37774698

RESUMEN

The role of tumor mutational burden (TMB) in shaping tumor immunity is a key question that has not been addressable using genetically engineered mouse models (GEMMs) of lung cancer. To induce TMB in lung GEMMs, we expressed an ultra-mutator variant of DNA polymerase-E (POLE)P286R in lung epithelial cells. Introduction of PoleP286R allele into KrasG12D and KrasG12D; p53L/L (KP) models significantly increase their TMB. Immunogenicity and sensitivity to immune checkpoint blockade (ICB) induced by Pole is partially dependent on p53. Corroborating these observations, survival of NSCLC patients whose tumors have TP53truncating mutations is shorter than those with TP53WT with immunotherapy. Immune resistance is in part through reduced antigen presentation and in part due to mutational heterogeneity. Total STING protein levels are elevated in Pole mutated KP tumors creating a vulnerability. A stable polyvalent STING agonist or p53 induction increases sensitivity to immunotherapy offering therapeutic options in these polyclonal tumors.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Ratones , Animales , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína p53 Supresora de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/terapia , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Mutación
8.
J Clin Invest ; 132(12)2022 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-35703180

RESUMEN

FOXA2 encodes a transcription factor mutated in 10% of endometrial cancers (ECs), with a higher mutation rate in aggressive variants. FOXA2 has essential roles in embryonic and uterine development. However, FOXA2's role in EC is incompletely understood. Functional investigations using human and mouse EC cell lines revealed that FOXA2 controls endometrial epithelial gene expression programs regulating cell proliferation, adhesion, and endometrial-epithelial transition. In live animals, conditional inactivation of Foxa2 or Pten alone in endometrial epithelium did not result in ECs, but simultaneous inactivation of both genes resulted in lethal ECs with complete penetrance, establishing potent synergism between Foxa2 and PI3K signaling. Studies in tumor-derived cell lines and organoids highlighted additional invasion and cell growth phenotypes associated with malignant transformation and identified key mediators, including Myc and Cdh1. Transcriptome and cistrome analyses revealed that FOXA2 broadly controls gene expression programs through modification of enhancer activity in addition to regulating specific target genes, rationalizing its tumor suppressor functions. By integrating results from our cell lines, organoids, animal models, and patient data, our findings demonstrated that FOXA2 is an endometrial tumor suppressor associated with aggressive disease and with shared commonalities among its roles in endometrial function and carcinogenesis.


Asunto(s)
Neoplasias Endometriales , Transición Epitelial-Mesenquimal , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Neoplasias Endometriales/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Ratones , Fosfatidilinositol 3-Quinasas
9.
Am J Surg Pathol ; 46(3): 404-414, 2022 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-34545858

RESUMEN

The diagnosis of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (AH/EIN) remains challenging and subjective in some cases, with variable histologic criteria and differences of opinion among gynecologic pathologists, potentially leading to under/overtreatment. There has been growing interest in the use of specific immunohistochemical markers as adjuncts in AH/EIN diagnosis. For example, the World Health Organization 2020 Classification specifies that loss of Pten, Pax2, or mismatch repair proteins are desirable diagnostic criteria. Other markers, most notably ß-catenin and Arid1a, are also aberrantly expressed in some AH/EIN. However, the performance of some markers individually-and more importantly as a group-has not been rigorously explored, raising questions as to which marker(s) or combination(s) is the most effective in practice. Formalin-fixed paraffin-embedded tissue sections from AH/EIN cases (n=111) were analyzed by immunohistochemistry for 6 markers: Pax2, Pten, Mlh1, ß-catenin, Arid1a, and p53. Aberrant expression was tabulated for each case and marker. An additional set of normal endometria (n=79) was also analyzed to define optimal diagnostic criteria for marker aberrance. The performance characteristics of each marker, the entire panel, and subsets thereof were quantitatively and statistically analyzed. In order of number of cases detected, the most frequently aberrant markers in AH/EIN were Pax2 (81.1% of cases), Pten (50.5%), ß-catenin (47.7%), Arid1a (7.2%), Mlh1 (4.5%), and p53 (2.7%). The majority of cases showed aberrant expression of ≥2 markers. All 6 markers together identified 92.8% of cases. Arid1a, Mlh1, and p53 were robust and readily scored markers, but all cases showing aberrant expression of these 3 markers were also detected by Pax2, Pten, or ß-catenin. A focused panel of only 3 markers (Pax2, Pten, and ß-catenin) showed optimal performance characteristics as a diagnostic adjunct in the histopathologic diagnosis of AH/EIN. Use of this panel is practicable and robust, with at least 1 of the 3 markers being aberrant in 92.8% of AH/EIN.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Hiperplasia Endometrial/diagnóstico , Homólogo 1 de la Proteína MutL/metabolismo , Factor de Transcripción PAX2/metabolismo , Fosfohidrolasa PTEN/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , beta Catenina/metabolismo , Biomarcadores/metabolismo , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patología , Femenino , Humanos , Inmunohistoquímica , Lesiones Precancerosas/diagnóstico , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología
10.
JCI Insight ; 5(14)2020 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-32699191

RESUMEN

Cancer is instigated by mutator phenotypes, including deficient mismatch repair and p53-associated chromosomal instability. More recently, a distinct class of cancers was identified with unusually high mutational loads due to heterozygous amino acid substitutions (most commonly P286R) in the proofreading domain of DNA polymerase ε, the leading strand replicase encoded by POLE. Immunotherapy has revolutionized cancer treatment, but new model systems are needed to recapitulate high mutational burdens characterizing human cancers and permit study of mechanisms underlying clinical responses. Here, we show that activation of a conditional LSL-PoleP286R allele in endometrium is sufficient to elicit in all animals endometrial cancers closely resembling their human counterparts, including very high mutational burden. Diverse investigations uncovered potentially novel aspects of Pole-driven tumorigenesis, including secondary p53 mutations associated with tetraploidy, and cooperation with defective mismatch repair through inactivation of Msh2. Most significantly, there were robust antitumor immune responses with increased T cell infiltrates, accelerated tumor growth following T cell depletion, and unfailing clinical regression following immune checkpoint therapy. This model predicts that human POLE-driven cancers will prove consistently responsive to immune checkpoint blockade. Furthermore, this is a robust and efficient approach to recapitulate in mice the high mutational burdens and immune responses characterizing human cancers.


Asunto(s)
ADN Polimerasa II/genética , Neoplasias Endometriales/genética , Inmunoterapia , Mutación/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Animales , Carcinogénesis/genética , Carcinogénesis/inmunología , Inestabilidad Cromosómica/genética , Inestabilidad Cromosómica/inmunología , Reparación de la Incompatibilidad de ADN/genética , Reparación de la Incompatibilidad de ADN/inmunología , Modelos Animales de Enfermedad , Neoplasias Endometriales/inmunología , Neoplasias Endometriales/patología , Neoplasias Endometriales/terapia , Endometrio/efectos de los fármacos , Endometrio/inmunología , Endometrio/metabolismo , Endometrio/patología , Femenino , Ratones , Fenotipo
11.
J Neuropathol Exp Neurol ; 68(6): 626-32, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19458547

RESUMEN

Hemangiomas are angiogenesis-dependent benign vascular tumors that can rupture and cause intracranial hemorrhages. We previously showed that the transcription factor homeobox A5 (HoxA5), which is absent in activated angiogenic endothelial cells can block angiogenesis. Here, we investigated whether restoring expression of HoxA5 blocks hemangioma growth by transplanting mouse hemangioendothelioma endothelial cells (EOMA) or HoxA5-expressing EOMA cells into the brains of mice. The EOMA cells induced brain hemangiomas characterized by large cystlike spaces lined by thin walls of endothelial cells surrounded by scant smooth muscle cells. When HoxA5-expressing EOMA cells were injected, lesion volumes were reduced between 5- and 20-fold compared with the EOMA control group (p < 0.05). Restoration of HoxA5 was associated with increased thrombospondin-2, which inhibits angiogenesis and reduced hypoxia-inducible factor 1alpha expression. These data suggest that restoring HoxA5 can attenuate experimental brain hemangioma development.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatología , Regulación Neoplásica de la Expresión Génica , Hemangioma/metabolismo , Hemangioma/fisiopatología , Proteínas de Homeodominio/metabolismo , Fosfoproteínas/metabolismo , Actinas/metabolismo , Animales , Neoplasias Encefálicas/etiología , Neoplasias Encefálicas/cirugía , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/trasplante , Regulación Neoplásica de la Expresión Génica/genética , Hemangioma/etiología , Hemangioma/cirugía , Proteínas de Homeodominio/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lectinas , Masculino , Ratones , Fosfoproteínas/genética , ARN Mensajero/metabolismo , Trombospondinas/metabolismo , Factores de Tiempo , Factores de Transcripción , Transfección/métodos
12.
J Clin Invest ; 128(9): 4179-4191, 2018 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-30124468

RESUMEN

Mutations underlie all cancers, and their identification and study are the foundation of cancer biology. We describe what we believe to be a novel approach to mutagenesis and cancer studies based on the DNA polymerase ε (POLE) ultramutator phenotype recently described in human cancers, in which a single amino acid substitution (most commonly P286R) in the proofreading domain results in error-prone DNA replication. We engineered a conditional PoleP286R allele in mice. PoleP286R/+ embryonic fibroblasts exhibited a striking mutator phenotype and immortalized more efficiently. PoleP286R/+ mice were born at Mendelian ratios but rapidly developed lethal cancers of diverse lineages, yielding the most cancer-prone monoallelic model described to date, to our knowledge. Comprehensive whole-genome sequencing analyses showed that the cancers were driven by high base substitution rates in the range of human cancers, overcoming a major limitation of previous murine cancer models. These data establish polymerase-mediated ultramutagenesis as an efficient in vivo approach for the generation of diverse animal cancer models that recapitulate the high mutational loads inherent to human cancers.


Asunto(s)
ADN Polimerasa II/metabolismo , Mutagénesis , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Sustitución de Aminoácidos , Animales , Linaje de la Célula/genética , ADN Polimerasa II/genética , Femenino , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Modelos Genéticos , Neoplasias Experimentales/patología , Proteínas de Unión a Poli-ADP-Ribosa/genética , Secuenciación Completa del Genoma
13.
Cancer Res ; 65(12): 5070-5, 2005 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-15958550

RESUMEN

Meningiomas constitute the second most common central nervous system tumor, and yet relatively little is known about the molecular events that are important for the pathogenesis and malignant progression of these tumors. We have used serial analysis of gene expression to compare the transcriptomes of nonneoplastic meninges and meningiomas of all malignancy grades. A novel finding from this screen is the induction of three components of the Notch signaling pathway: the transcription factor, hairy and enhancer of Split1 (HES1) and two members of the Groucho/transducin-like enhancer of Split family of corepressors, TLE2 and TLE3. TLE corepressors interact and modulate the activity of a wide range of transcriptional regulatory systems, one of which is HES1. We have shown that the transcript and protein levels of HES1, the Notch2 and Notch1 receptors and the Jagged1 ligand are induced in meningiomas of all grades, whereas induction of TLE2 and TLE3 occurs specifically in higher-grade meningiomas. Meningioma cell lines express components of the Notch signaling pathway and an inhibitor of this pathway suppresses meningioma cell survival. These results suggest that deregulated expression of the Notch pathway is a critical event in meningioma pathogenesis and that modulation of this and potentially other signaling pathways by TLE corepressors leads to a more malignant phenotype.


Asunto(s)
Neoplasias Meníngeas/genética , Meningioma/genética , Receptores de Superficie Celular/fisiología , Factores de Transcripción/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Unión al Calcio , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Ligandos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/parasitología , Meningioma/metabolismo , Meningioma/patología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , Receptor Notch1 , Receptor Notch2 , Receptores de Superficie Celular/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteínas Serrate-Jagged , Transducción de Señal , Factor de Transcripción HES-1 , Factores de Transcripción/genética
14.
Methods Mol Biol ; 1463: 139-154, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27734354

RESUMEN

The precise identity of spermatogonial stem cells-the germline stem cell of the adult testis-remains a controversial topic. Technical limitations have included the lack of specific markers and methods for lineage tracing of Asingle spermatogonia and their subsets. Immunolocalization of proteins in tissue sections has been a standard tool for the in situ identification and visualization of rare cellular subsets. However, these studies are limited by the need for faithful and reliable protein markers to define these cell types, as well as the availability of specific antibodies to these markers. Here we describe the use of a monoclonal antibody to Pax7 as a means to detect spermatogonial stem cells (SSCs) both in tissue sections and in intact seminiferous tubules. Furthermore, we describe methods for lineage tracing as an alternative method to visualize Pax7+ spermatogonial stem cells and their progeny.


Asunto(s)
Factor de Transcripción PAX7/metabolismo , Espermatogonias/citología , Células Madre/citología , Animales , Diferenciación Celular , Rastreo Celular , Masculino , Ratones , Espermatogénesis , Espermatogonias/metabolismo , Células Madre/metabolismo
15.
Mol Biochem Parasitol ; 139(1): 15-24, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15610815

RESUMEN

Nuclear export and import of proteins and RNAs is a regulated process that permits the control of protein expression during cell development and differentiation. In all eukaryotic organisms transport of proteins to specific cellular compartments requires specific signaling sequences. Proteins that shuttle between nucleus and cytoplasm bear nuclear localization signals (NLS) and/or nuclear export signals (NES) and some of them can carry mRNAs, as part of shuttling ribonucleoprotein complexes. In this work we describe in the protozoan parasite Trypanosoma cruzi, a CRM1/exportin1 nuclear export factor named TcCRM1. This protein contains the conserved central region (CCR) that interacts with NES sequences present within cargo molecules, and the Cys residue involved in covalent binding to the Streptomyces metabolite leptomycin B (LMB). By subcellular fractionation we show that TcCRM1, a protein of about 117 kDa, has nuclear localization. We also demonstrate that LMB inhibits the replication of T. cruzi in a dose-dependent manner. In situ hybridization experiments performed with a Texas red-coupled oligo(dT) probe revealed that LMB produced a partial short-term accumulation of a poly(A)+RNA subset in the nucleus. Some mRNAs such as HSP70, TcUBP2/1 and TcPABP1 are reduced or disappeared from the cytoplasm of LMB treated cells. In sharp contrast with metazoans, no effect was observed on two U snRNAs subcellular localization, implying that a different export route might exist for these RNAs in trypanosomes.


Asunto(s)
Carioferinas/metabolismo , Transporte de ARN , ARN Protozoario/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Trypanosoma cruzi/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Antiprotozoarios/metabolismo , Antiprotozoarios/farmacología , División Celular , Núcleo Celular/metabolismo , Clonación Molecular , Secuencia Conservada/genética , ADN Protozoario/química , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Carioferinas/genética , Datos de Secuencia Molecular , Poli A/metabolismo , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Análisis de Secuencia de ADN , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiología , Proteína Exportina 1
16.
PLoS One ; 10(3): e0121720, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25821967

RESUMEN

HoxA5 is expressed in quiescent endothelial cells (EC), but absent in activated angiogenic EC. To examine the efficacy of targeting HoxA5 therapeutically to quell pathologic or tumor angiogenesis, we generated an inducible, transgenic mouse model of sustained HoxA5 expression in ECs. During pathologic angiogenesis, sustained HoxA5 regulates expression several angiogenic effector molecules, notably increased expression of TSP-2 and reduced expression of VEGF, thus leading to inhibition of pathological angiogenesis in tissues. To evaluate if this impressive reduction of vascularization could also impact tumor angiogenesis, HoxA5 mice were bred with a mouse model of de novo squamous carcinogenesis, e.g., K14-HPV16 mice. Activation of EC-HoxA5 significantly reduced infiltration by mast cells into neoplastic skin, an early hallmark of progression to dysplasia, reduced angiogenic vasculature, and blunted characteristics of tumor progression. To evaluate HoxA5 as a therapeutic, topical application of a HoxA5 transgene onto early neoplastic skin of K14-HPV16 mice similarly resulted in a significant impairment of angiogenic vasculature and progression to dysplasia to a similar extent as observed with genetic delivery of HoxA5. Together these data indicate that HoxA5 represents a novel molecule for restricting pathological and tumorigenic angiogenesis.


Asunto(s)
Células Endoteliales/metabolismo , Células Endoteliales/patología , Proteínas de Homeodominio/genética , Neovascularización Patológica/genética , Fosfoproteínas/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Progresión de la Enfermedad , Células Endoteliales/virología , Papillomavirus Humano 16 , Ratones , Ratones Transgénicos/genética , Procesos Neoplásicos , Neovascularización Patológica/patología , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Lesiones Precancerosas/virología , Neoplasias Cutáneas/virología , Trombospondinas/genética , Factores de Transcripción , Transgenes/genética , Factor A de Crecimiento Endotelial Vascular/genética
17.
J Clin Invest ; 125(11): 4063-76, 2015 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-26413869

RESUMEN

Endometrial cancer is the most common gynecologic malignancy and the fourth most common malignancy in women. For most patients in whom the disease is confined to the uterus, treatment results in successful remission; however, there are no curative treatments for tumors that have progressed beyond the uterus. The serine/threonine kinase LKB1 has been identified as a potent suppressor of uterine cancer, but the biological modes of action of LKB1 in this context remain incompletely understood. Here, we have shown that LKB1 suppresses tumor progression by altering gene expression in the tumor microenvironment. We determined that LKB1 inactivation results in abnormal, cell-autonomous production of the inflammatory cytokine chemokine (C-C motif) ligand 2 (CCL2) within tumors, which leads to increased recruitment of macrophages with prominent tumor-promoting activities. Inactivation of Ccl2 in an Lkb1-driven mouse model of endometrial cancer slowed tumor progression and increased survival. In human primary endometrial cancers, loss of LKB1 protein was strongly associated with increased CCL2 expression by tumor cells as well as increased macrophage density in the tumor microenvironment. These data demonstrate that CCL2 is a potent effector of LKB1 loss in endometrial cancer, creating potential avenues for therapeutic opportunities.


Asunto(s)
Adenocarcinoma/patología , Quimiocina CCL2/fisiología , Neoplasias Endometriales/patología , Macrófagos/inmunología , Proteínas de Neoplasias/fisiología , Proteínas Serina-Treonina Quinasas/deficiencia , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/fisiología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Animales , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/sangre , Ácido Clodrónico/farmacología , Ácido Clodrónico/uso terapéutico , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Neoplasias Endometriales/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Macrófagos/efectos de los fármacos , Ratones , Invasividad Neoplásica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/sangre , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , Organismos Libres de Patógenos Específicos , Transcripción Genética , Microambiente Tumoral
18.
Adv Wound Care (New Rochelle) ; 3(10): 605-613, 2014 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-25302136

RESUMEN

Objective: Homeobox (HOX) transcription factors coordinate gene expression in wound repair and angiogenesis. Previous studies have shown that gene transfer of HoxA3 to wounds of diabetic mice accelerates wound healing, increasing angiogenesis and keratinocyte migration. In this study, we examined whether HoxA3 can also improve angiogenesis, epidermal integrity, and viability of composite skin grafts. Approach: To determine the effects of HoxA3 on composite skin grafts, we constructed bilayered composite grafts incorporating fibroblasts engineered to constitutively secrete HoxA3. We then transplanted these composite grafts in vivo. Results: The composite grafts produced a stratified epidermal layer after seventeen days in culture and following transplantation in vivo, these grafts exhibit normal epidermal differentiation and reduced contraction compared to controls. In addition, HoxA3 grafts showed increased angiogenesis. Quantitative polymerase chain reaction (PCR) analyses of HoxA3 graft tissue reveal an increase in the downstream HoxA3 target genes MMP-14 and uPAR expression, as well as a reduction in CCL-2 and CxCl-12. Innovation: Expression of secreted HoxA3 in composite grafts represents a comprehensive approach that targets both keratinocytes and endothelial cells to promote epidermal proliferation and angiogenesis. Conclusion: Secreted HoxA3 improves angiogenesis, reduces expression of inflammatory mediators, and prolongs composite skin graft integrity.

19.
J Clin Invest ; 124(9): 3929-44, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25133429

RESUMEN

Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of A(single) spermatogonia in mice. PAX7+ cells were present in the testis at birth. Compared with the adult testis, PAX7+ cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that PAX7+ spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, PAX7+ spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, PAX7+ spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the PAX7+ subset of A(single) spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germline injury, such as occurs after cancer therapy.


Asunto(s)
Factor de Transcripción PAX7/fisiología , Células Madre/química , Testículo/citología , Animales , Infertilidad Masculina/etiología , Masculino , Ratones , Factor de Transcripción PAX7/análisis , Espermatogénesis , Espermatogonias/fisiología , Testículo/metabolismo
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