RESUMEN
Basal cells are adult stem cells in the airway epithelium and regenerate differentiated cell populations, including the mucosecretory and ciliated cells that enact mucociliary clearance. Human basal cells can proliferate and produce differentiated epithelium in vitro. However, studies of airway epithelial differentiation mostly rely on immunohistochemical or immunofluorescence-based staining approaches, meaning that a dynamic approach is lacking, and quantitative data is limited. Here, we use a lentiviral reporter gene approach to transduce primary human basal cells with bioluminescence reporter constructs to monitor airway epithelial differentiation longitudinally. We generated three constructs driven by promoter sequences from the TP63, MUC5AC and FOXJ1 genes to quantitatively assess basal cell, mucosecretory cell and ciliated cell abundance, respectively. We validated these constructs by tracking differentiation of basal cells in air-liquid interface and organoid ('bronchosphere') cultures. Transduced cells also responded appropriately to stimulation with interleukin 13 (IL-13; to increase mucosecretory differentiation and mucus production) and IL-6 (to increase ciliated cell differentiation). These constructs represent a new tool for monitoring airway epithelial cell differentiation in primary epithelial and/or induced pluripotent stem cell (iPSC) cell cultures.
RESUMEN
The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E2), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER- cells. However, the presence of LRH-1 protein in ER- cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER- breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER- compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E2, showed increased mRNA stability in ER- versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E2 treatment, this effect mediated by ERα. Our data demonstrates that in ER- cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER- cells as well as ER- tumors suggests a possible role in the development of ER- tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER- and ER+ breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Estabilidad Proteica/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Isoformas de Proteínas/genética , Estabilidad del ARN/genética , ARN Mensajero/metabolismoRESUMEN
The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells, and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium was similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony formation ability, sustained in vitro growth, and outcompeted adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states.
RESUMEN
It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.
Asunto(s)
Proteína BRCA1/genética , Transformación Celular Neoplásica/genética , Neoplasias Mamarias Experimentales/genética , Fenobarbital/metabolismo , Análisis de la Célula Individual/métodos , Células Madre/patología , Animales , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Mutación , Células Madre/fisiología , Microambiente Tumoral/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Patients diagnosed with lung squamous cell carcinoma (LUSC) have limited targeted therapies. We report here the identification and characterisation of BCL11A, as a LUSC oncogene. Analysis of cancer genomics datasets revealed BCL11A to be upregulated in LUSC but not in lung adenocarcinoma (LUAD). Experimentally we demonstrate that non-physiological levels of BCL11A in vitro and in vivo promote squamous-like phenotypes, while its knockdown abolishes xenograft tumour formation. At the molecular level we found that BCL11A is transcriptionally regulated by SOX2 and is required for its oncogenic functions. Furthermore, we show that BCL11A and SOX2 regulate the expression of several transcription factors, including SETD8. We demonstrate that shRNA-mediated or pharmacological inhibition of SETD8 selectively inhibits LUSC growth. Collectively, our study indicates that BCL11A is integral to LUSC pathology and highlights the disruption of the BCL11A-SOX2 transcriptional programme as a novel candidate for drug development.
Asunto(s)
Carcinoma de Células Escamosas/genética , Proteínas Portadoras/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción SOXB1/metabolismo , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/genética , Técnicas de Silenciamiento del Gen , Sitios Genéticos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Oncogenes , Organoides/patología , Unión Proteica , Proteínas RepresorasRESUMEN
Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2-ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success.Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples.We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors.Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype.Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/tratamiento farmacológico , Progresión de la Enfermedad , Losartán/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Receptor de Angiotensina Tipo 1/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Biopsia , Carcinogénesis/metabolismo , Carcinoma Intraductal no Infiltrante/inducido químicamente , Carcinoma Intraductal no Infiltrante/inmunología , Carcinoma Intraductal no Infiltrante/patología , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Inmunohistoquímica , Interleucina-6/metabolismo , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Acetato de Medroxiprogesterona/toxicidad , Ratones , Invasividad Neoplásica , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , Sistema Renina-Angiotensina/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
The cytokine Tumor Necrosis Factor-α is critical to Estrogen Receptor positive breast cancer pathology, stimulating estrogen-biosynthesis pathways and preventing the differentiation of estrogen-producing fibroblasts. High concentrations of TNFα are detected in the tumor microenvironment, and infiltrating immune cells are thought to be a major source. This study identifies that TNFα is also a tumor-derived factor, expressed in ER+ tumour epithelial cells and regulated by 17-ß-estradiol (E2). Treatment of MCF-7, T47D and ZR-75 breast cancer cells with E2 increased TNFα mRNA and protein expression and secretion. This effect was mitigated with the use of ERα inhibitors 4-hydroy-tamoxifen and ICI-182780, indicating that E2-mediated TNFα induction was via the actions of ERα. Chromatin immunoprecipitation reveals ERα binding to the TNFα promoter upon stimulation with E2. This study demonstrates for the first time a positive feedback loop between estradiol and TNFα, critical in maintaining high levels of the hormone within the ER+ breast tumour microenvironment.
Asunto(s)
Células Epiteliales/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Estradiol/análogos & derivados , Estradiol/metabolismo , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Retroalimentación Fisiológica , Femenino , Fulvestrant , Humanos , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/metabolismo , Transducción de Señal , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Factor de Necrosis Tumoral alfa/agonistas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor that belongs to the NR5A subgroup of nuclear receptors. LRH-1 induces key genes to regulate metabolic process, ovarian function, cancer cell proliferation, and steroidogenesis. In the breast, LRH-1 modulates and synergizes with endogenous estrogen signaling to promote breast cancer cell proliferation. We used small interfering RNA knockdown strategies to deplete LRH-1 in breast cancer cells and followed with microarray analysis to identify LRH-1-dependent mechanisms. We identified key genes involved in TGF-ß signaling to be highly responsive to LRH-1 knockdown. This relationship was validated in 2 breast cancer cell lines overexpressing LRH-1 in vitro and in a novel transgenic mouse with targeted LRH-1 overexpression in mammary epithelial cells. Notably, TGF-ß signaling was activated in LRH-1-overexpressing breast cancer cells and mouse mammary glands. Further analyses of mammary gross morphology revealed a significant reduction in mammary lateral budding after LRH-1 overexpression. These findings suggest that the altered mammary morphogenesis in LRH-1 transgenic animals is mediated via enhanced TGF-ß expression. The regulation of TGF-ß isoforms and SMAD2/3-mediated downstream signaling by LRH-1 also implicates a potential contribution of LRH-1 in breast cancer. Collectively, these data demonstrate that LRH-1 regulates TGF-ß expression and downstream signaling in mouse mammary glands.
Asunto(s)
Neoplasias de la Mama/metabolismo , Glándulas Mamarias Animales/metabolismo , Morfogénesis , Receptores Citoplasmáticos y Nucleares/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Regulación hacia Arriba , Animales , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Células MCF-7 , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/patología , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteínas Smad Reguladas por Receptores/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Liver Receptor Homolog-1 (LRH-1; NR5A2) belongs to the orphan nuclear receptor superfamily, and plays vital roles in early development, cholesterol homeostasis, steroidogenesis and certain diseases, including cancer. It is expressed in embryonic stem cells, adult liver, intestine, pancreas and ovary. It binds to DNA as a monomer and is regulated by various ligand-dependent and -independent mechanisms. Recent work identified synthetic ligands for LRH-1; such compounds may yield useful therapeutics for a range of pathologic conditions associated with aberrant expression and activity of LRH-1.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/agonistas , Animales , Colesterol/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Femenino , Humanos , Masculino , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Fosfolípidos/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
BACKGROUND: Liver Receptor Homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that is over-expressed in cancers in tissues such as the breast, colon and pancreas. LRH-1 plays important roles in embryonic development, steroidogenesis and cholesterol homeostasis. In tumor cells, LRH-1 induces proliferation and cell cycle progression. High LRH-1 expression is demonstrated in breast cancers, positively correlating with ERα status and aromatase activity. LRH-1 dependent cellular mechanisms in breast cancer epithelial cells are poorly defined. Hence in the present study we investigated the actions of LRH-1 in estrogen receptor α (ERα) positive breast cancer cells. RESULTS: The study aimed to investigate LRH-1 dependent mechanisms that promote breast cancer proliferation. We identified that LRH-1 regulated the expression of Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) in MCF-7 and MDA-MB-231 cells. Over-expression of LRH-1 increased GREB1 mRNA levels while knockdown of LRH-1 reduced its expression. GREB1 is a well characterised ERα target gene, with three estrogen response elements (ERE) located on its promoter. Chromatin immunoprecipitation studies provided evidence of the co-localisation of LRH-1 and ERα at all three EREs. With electrophoretic mobility shift assays, we demonstrated direct binding of LRH-1 to EREs located on GREB1 and Trefoil Factor 1 (TFF1, pS2) promoters. LRH-1 and ERα co-operatively activated transcription of ERE luciferase reporter constructs suggesting an overlap in regulation of target genes in breast cancer cells. Over-expression of LRH-1 resulted in an increase in cell proliferation. This effect was more pronounced with estradiol treatment. In the presence of ICI 182,780, an ERα antagonist, LRH-1 still induced proliferation. CONCLUSIONS: We conclude that in ER-positive breast cancer cells, LRH-1 promotes cell proliferation by enhancing ERα mediated transcription of target genes such as GREB-1. Collectively these findings indicate the importance of LRH-1 in the progression of hormone-dependent breast cancer and implicate LRH-1 as a potential avenue for drug development.
Asunto(s)
Neoplasias de la Mama/patología , Proliferación Celular , Receptor alfa de Estrógeno/fisiología , Proteínas de Neoplasias/genética , Receptores Nucleares Huérfanos/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Elementos de Respuesta , Transcripción GenéticaRESUMEN
BACKGROUND: Granulosa cell tumors (GCT) of the ovary often express aromatase and synthesize estrogen, which in turn may influence their progression. Recently a specific point mutation (C134W) in the FOXL2 protein was identified in >94% of adult-type GCT and it is likely to contribute to their development. A number of genes are known to be regulated by FOXL2, including aromatase/CYP19A1, but it is unclear which are direct targets and whether the C134W mutation alters their regulation. Recently, it has been reported that FOXL2 forms a complex with steroidogenic factor 1 (SF-1) which is a known regulator of aromatase in granulosa cells. METHODOLOGY/PRINCIPAL FINDINGS: In this work, the human GCT-derived cell lines KGN and COV434 were heterozygous and wildtype for the FOXL2:C134W mutation, respectively. KGN had abundant FOXL2 mRNA expression but it was not expressed in COV434. Expression of exogenous FOXL2:C134W in COV434 cells induced higher expression of a luciferase reporter for the ovarian specific aromatase promoter, promoter II (PII) (-516bp) than expression of wildtype FOXL2, but did not alter induction of a similar reporter for the steroidogenic acute regulatory protein (StAR) promoter (-1300bp). Co-immunoprecipitation confirmed that FOXL2 bound SF-1 and that it also bound its homologue, liver receptor homologue 1 (LRH-1), however, the C134W mutation did not alter these interactions or induce a selective binding of the proteins. A highly conserved putative binding site for FOXL2 was identified in PII. FOXL2 was demonstrated to bind the site by electrophoretic mobility shift assays (EMSA) and site-directed mutagenesis of this element blocked its differential induction by wildtype FOXL2 and FOXL2:C134W. CONCLUSIONS/SIGNIFICANCE: These findings suggest that aromatase is a direct target of FOXL2:C134W in adult-type GCT via a single distinctive and highly conserved binding site in PII and therefore provide insight into the pathogenic mechanism of this mutation.