RESUMEN
Recent literature highlights the importance of pro-inflammatory cytokines in the biology of breast cancer stem cells (CSCs), unraveling differences with respect to their normal counterparts. Expansion of mammospheres (MS) is a valuable tool for the in vitro study of normal and cancer mammary gland stem cells. Here, we expanded MSs from human breast cancer and normal mammary gland tissues, as well from tumorigenic (MCF7) and non-tumorigenic (MCF10) breast cell lines. We observed that agonists for the retinoid X receptor (6-OH-11-O-hydroxyphenanthrene), retinoic acid receptor (all-trans retinoic acid (RA)) and peroxisome proliferator-activated receptor (PPAR)-γ (pioglitazone (PGZ)), reduce the survival of MS generated from breast cancer tissues and MCF7 cells, but not from normal mammary gland or MCF10 cells. This phenomenon is paralleled by the hampering of pro-inflammatory Nuclear Factor-κB (NF-κB)/Interleukin-6 (IL6) axis that is hyperactive in breast cancer-derived MS. The hindrance of such pathway associates with the downregulation of MS regulatory genes (SLUG, Notch3, Jagged1) and with the upregulation of the differentiation markers estrogen receptor-α and keratin18. At variance, the PPARα agonist Wy14643 promotes MS formation, upregulating NF-κB/IL6 axis and MS regulatory genes. These data reveal that nuclear receptors agonists (6-OH-11-O-hydroxyphenanthrene, RA, PGZ) reduce the inflammation dependent survival of breast CSCs and that PPARα agonist Wy14643 exerts opposite effects on this phenotype.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Células Madre Neoplásicas/citología , Fenantrenos/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazolidinedionas/farmacología , Tretinoina/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Interleucina-6/metabolismo , FN-kappa B/metabolismo , PPAR gamma/agonistas , PPAR gamma/metabolismo , Fenantrenos/química , Pioglitazona , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide/agonistas , Receptores X Retinoide/metabolismo , Tiazolidinedionas/química , Tretinoina/químicaRESUMEN
OBJECTIVES: This study aimed to isolate and characterize stem/progenitor cells, starting from normal airway epithelia, obtained from human adults. MATERIALS AND METHODS: Cultures of multicellular spheroids were obtained from human lung tissue specimens after mechanical and enzymatic digestion. Tissue-specific markers were detected on their cells by immunohistochemical and immunofluorescent techniques. Ultrastructural morphology of the spheroids (termed as bronchospheres) was evaluated by electron microscopy, gene expression analysis was performed by reverse transcription-polymerase chain reaction, and gene down-regulation was analysed by an RNA interference technique. RESULTS: Bronchospheres were found to be composed of cells with high expression of stem cell regulatory genes, which was not or was only weakly detectable in original tissues. Morphological analysis showed that bronchospheres were composed of mixed phenotype cells with type II alveolar and Clara cell features, highlighting their airway resident cell origin. In addition to displaying specific pulmonary and epithelial commitment, bronchospheres showed mesenchymal features. Silencing of the Slug gene, known to play a pivotal role in epithelial-mesenchymal transition processes and which was highly expressed in bronchospheres but not in original tissue, led bronchospheres to gain a differentiated bronchial/alveolar phenotype and to lose the stemness gene expression pattern. CONCLUSIONS: Ours is the first study to describe ex vivo expansion of stem/progenitor cells resident in human lung epithelia, and our results suggest that the epithelial-mesenchymal transition process, still active in a subset of airway cells, may regulate transit of stem/progenitor cells towards epithelial differentiation.
Asunto(s)
Separación Celular , Pulmón/citología , Células Madre/citología , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Diferenciación Celular , Femenino , Humanos , Inmunohistoquímica , Masculino , Mesodermo/citología , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Interferencia de ARN , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The p53 codon 72 polymorphism, which results in either an arginine or proline residue, plays a different role in vitro and in vivo in cell survival and drug resistance. We verified, in vitro, the impact of the arginine allele on cell survival under normoxia and hypoxia, and investigated in vivo the role of p53 codon 72 arginine homozygosity in the clinical outcome of advanced breast cancer patients. METHODS: Tumors at advanced stages grow in vivo in a hypoxic environment, and we mimicked such conditions in vitro using p53 null breast cancer cells transfected with either the arginine or proline allele. We also analyzed in vivo the p53 codon 72 genotype status of advanced breast cancer patients. RESULTS: In vitro transfection of the arginine allele induced higher cell death under normoxia, whereas cell death was greater in proline-transfected cells under hypoxia. The arginine allele upregulated BCRP-I, a hypoxia response gene, which increases drug resistance. Metastatic breast cancer patients homozygous for arginine had a significantly shorter time to progression and overall survival than those with heterozygous arginine/proline tumors. CONCLUSION: We provide a molecular explanation for the association of the arginine allele with tumor aggressiveness and treatment resistance in advanced breast cancer.
Asunto(s)
Alelos , Arginina/genética , Neoplasias de la Mama/genética , Codón/genética , Polimorfismo de Nucleótido Simple/genética , Proteína p53 Supresora de Tumor/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Adulto , Anciano , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Hipoxia/fisiopatología , Estimación de Kaplan-Meier , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Prolina/genética , Transfección , Regulación hacia ArribaRESUMEN
Basal-like breast carcinoma is an aggressive form of breast cancer, characterized by the absence of oestrogen receptor and HER2 expression, the presence of cytokeratin 5 and epidermal growth factor receptor expression, and by the up-regulation of stem cell regulatory genes. We show here that tumour tissues expressing high levels of SLUG mRNA show a basal-like breast carcinoma phenotype and that such tumours also express high levels of stem cell-regulatory genes, ie CD133, Bmi1. Further, we show that stem/progenitor cells, isolated from ductal breast carcinoma and from normal mammary gland as mammospheres, express SLUG, CD133, and Bmi1 mRNA and show a phenotype similar to that of basal-like breast carcinoma. We also report that SLUG expression in tumour tissues correlates with that of the hypoxia survival gene carbonic anhydrase IX. In this regard, we report that the exposure of SLUG-negative/luminal-like MCF-7 cells to a hypoxic environment promotes the onset of the basal-like breast carcinoma phenotype, together with up-regulation of the SLUG gene, which in turn blunts oestrogen receptor-alpha and boosts carbonic anhydrase IX gene expression. Finally, we show that SLUG expression promotes the invasiveness of MCF-7 cells exposed to hypoxia and sustains the in vivo aggressiveness of hypoxia-selected, MCF-7-derived cells in xenografts. These data indicate that SLUG gene expression is part of a hypoxia-induced genetic programme which sets up a basal/stem cell-like, aggressive phenotype in breast cancer cells.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/metabolismo , Factores de Transcripción/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/biosíntesis , Anhidrasas Carbónicas/genética , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Hipoxia de la Célula/genética , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Neoplásico/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
The preferential retention of the arginine allele at the p53 codon 72 locus is commonly observed in tumours from arginine/proline heterozygotes. Considering that cancer cells are harboured in a hypoxic environment in vivo, we here tested the hypothesis that the p53 codon 72 proline allele confers a survival disadvantage in presence of hypoxia. Here, we show that the transient transfection of the proline allele in p53 null cancer cells exposed to low oxygen tension or to the hypoxia-mimetic drug Desferoxamine induces a higher amount of cell death than the arginine allele. Accordingly, proline allele transiently transfected cell lines express lower levels of hypoxia pro-survival genes (HIF-1alpha, carbonic anhydrase IX, vascular endothelial growth factor, heme oxygenase-I, hepatocyte growth factor receptor, vascular endothelial growth factor receptor 2), compared to those transiently transfected with the arginine allele. Further, we report that the exposure of the arginine/proline heterozygote MCF-7 breast cancer cell line to cytotoxic concentration of Desferoxamine for several weeks, gives raise to hypoxia-resistant clones, carrying the arginine, but not the proline allele. These data indicate that the p53 codon 72 proline allele is less permissive for the growth of cancer cells in a hypoxic environment, and suggest that the preferential retention of the arginine allele in the tumour tissues of arginine/proline heterozygous patients may depend upon its lowered capacity to induce cell death in a hypoxic tumour environment.
Asunto(s)
Alelos , Apoptosis , Neoplasias de la Mama/genética , Hipoxia de la Célula , Codón , Genes p53 , Prolina/genética , Arginina/genética , Neoplasias de la Mama/patología , Femenino , HumanosRESUMEN
A common arginine to proline polymorphism is harboured at codon 72 of the human p53 gene. In this investigation, we found that fibroblasts and lymphocytes isolated from arginine allele homozygote centenarians and sexagenarians (Arg+) undergo an oxidative-stress-induced apoptosis at a higher extent than cells obtained from proline allele carriers (Pro+). At variance, the difference in apoptosis susceptibility between Arg+ and Pro+ is not significant when cells from 30-year-old people are studied. Further, we found that Arg+ and Pro+ cells from centenarians differ in the constitutive levels of p53 protein and p53/MDM2 complex, as well as in the levels of oxidative stress-induced p53/Bcl-xL complex and mitochondria-localised p53. Consistently, all these differences are less evident in cells from 30-year-old people. Finally, we investigated the in vivo functional relevance of the p53 codon 72 genotype in a group of old patients (66-99 years of age) affected by acute myocardial ischaemia, a clinical condition in which in vivo cell death occurs. We found that Arg+ patients show increased levels of Troponin I and CK-MB, two serum markers that correlate with the extent of the ischaemic damage in comparison to Pro+ patients. In conclusion, these data suggest that p53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia.
Asunto(s)
Apoptosis , Codón , Genes p53 , Isquemia , Proteína p53 Supresora de Tumor/fisiología , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Alelos , Arginina , Western Blotting , Muerte Celular , Creatina Quinasa/sangre , Forma MB de la Creatina-Quinasa , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/metabolismo , Citometría de Flujo , Genotipo , Homocigoto , Humanos , Inmunoprecipitación , Isoenzimas/sangre , Leucocitos/metabolismo , Linfocitos/metabolismo , Masculino , Potenciales de la Membrana , Microscopía Fluorescente , Persona de Mediana Edad , Isquemia Miocárdica/patología , Estrés Oxidativo , Polimorfismo Genético , Prolina , Proteínas Proto-Oncogénicas c-bcl-2 , Análisis de Regresión , Serina/química , Factores de Tiempo , Transfección , Troponina I/sangre , Proteína bcl-XRESUMEN
The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytotoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapy drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.