RESUMEN
Retinoid X receptors (RXRs) are obligate partners for several other nuclear receptors, and they play a key role in several signaling processes. Despite being a promiscuous heterodimer partner, this nuclear receptor is a target of therapeutic intervention through activation using selective RXR agonists (rexinoids). Agonist binding to RXR initiates a large conformational change in the receptor that allows for coactivator recruitment to its surface and enhanced transcription. Here we reveal the structural and dynamical changes produced when a coactivator peptide binds to the human RXRα ligand binding domain containing two clinically relevant rexinoids, Targretin and 9-cis-UAB30. Our results show that the structural changes are very similar for each rexinoid and similar to those for the pan-agonist 9-cis-retinoic acid. The four structural changes involve key residues on helix 3, helix 4, and helix 11 that move from a solvent-exposed environment to one that interacts extensively with helix 12. Hydrogen-deuterium exchange mass spectrometry reveals that the dynamics of helices 3, 11, and 12 are significantly decreased when the two rexinoids are bound to the receptor. When the pan-agonist 9-cis-retinoic acid is bound to the receptor, only the dynamics of helices 3 and 11 are reduced. The four structural changes are conserved in all x-ray structures of the RXR ligand-binding domain in the presence of agonist and coactivator peptide. They serve as hallmarks for how RXR changes conformation and dynamics in the presence of agonist and coactivator to initiate signaling.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Naftalenos/metabolismo , Coactivador 2 del Receptor Nuclear/metabolismo , Receptor alfa X Retinoide/metabolismo , Tetrahidronaftalenos/metabolismo , Alitretinoína , Secuencia de Aminoácidos , Bexaroteno , Sitios de Unión , Cristalografía por Rayos X , Ácidos Grasos Insaturados/química , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Naftalenos/química , Coactivador 2 del Receptor Nuclear/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptor alfa X Retinoide/química , Tetrahidronaftalenos/química , Tretinoina/química , Tretinoina/metabolismoRESUMEN
(2E,4E,6Z,8Z)-8-(3',4'-Dihydro-1'(2H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,3,6-octatrienoinic acid, 9cUAB30, is a selective rexinoid for the retinoid X nuclear receptors (RXR). 9cUAB30 displays substantial chemopreventive capacity with little toxicity and is being translated to the clinic as a novel cancer prevention agent. To improve on the potency of 9cUAB30, we synthesized 4-methyl analogs of 9cUAB30, which introduced chirality at the 4-position of the tetralone ring. The syntheses and biological evaluations of the racemic homolog and enantiomers are reported. We demonstrate that the S-enantiomer is the most potent and least toxic even though these enantiomers bind in a similar conformation in the ligand binding domain of RXR.
Asunto(s)
Neoplasias/prevención & control , Neoplasias/terapia , Receptores X Retinoide/metabolismo , Retinoides/química , Humanos , Factor 4 Similar a Kruppel , Ligandos , Conformación Molecular , Retinoides/metabolismoRESUMEN
The use of in vitro, engineered surrogates in the field of cancer research is of interest for studies involving mechanisms of growth and metastasis, and response to therapeutic intervention. While biomimetic surrogates better model human disease, their complex composition and dimensionality make them challenging to evaluate in a real-time manner. This feature has hindered the broad implementation of these models, particularly in drug discovery. Herein, several methods and approaches for the real-time, non-invasive analysis of cell growth and response to treatment in tissue-engineered, three-dimensional models of breast cancer are presented. The tissue-engineered surrogates used to demonstrate these methods consist of breast cancer epithelial cells and fibroblasts within a three dimensional volume of extracellular matrix and are continuously perfused with nutrients via a bioreactor system. Growth of the surrogates over time was measured using optical in vivo (IVIS) imaging. Morphologic changes in specific cell populations were evaluated by multi-photon confocal microscopy. Response of the surrogates to treatment with paclitaxel was measured by optical imaging and by analysis of lactate dehydrogenase and caspase-cleaved cytokeratin 18 in the perfused medium. Each method described can be repeatedly performed during culture, allowing for real-time, longitudinal analysis of cell populations within engineered tumor models.
Asunto(s)
Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Animales , Reactores Biológicos , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Ensayos de Selección de Medicamentos Antitumorales/métodos , Diseño de Equipo , Matriz Extracelular/patología , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Queratina-18/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Mediciones Luminiscentes/métodos , Ratones , Microscopía Confocal , Paclitaxel/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes, KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions.
Asunto(s)
Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/biosíntesis , Células Epiteliales/patología , Queratinocitos/citología , Piel/patología , Factores de Transcripción/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Cruzamientos Genéticos , Cartilla de ADN , Doxorrubicina/farmacología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , TransfecciónRESUMEN
The hedgehog pathway regulates epithelial-mesenchymal interactions, differentiation, proliferation and survival during development. Stimulation of hedgehog signaling induces carcinogenesis or promotes cell survival in cancers of multiple organs. Using real-time, quantitative PCR, laser capture microdissection, and immunohistochemistry, distinctive patterns of expression of the hedgehog pathway members patched 1 (PTCH1), smoothened, GLI1, GLI2 and the 3 hedgehog ligands were identified for epithelial cells and stromal fibroblasts in benign breast and breast cancer. Hedgehog ligands were expressed at higher levels in some cancer epithelial cell lines compared to noncancerous epithelial cells. Correspondingly, expression of GLI1, a transcription factor and transcriptional product of hedgehog signaling, was increased 8-fold in cancer epithelial cell lines; however, PTCH1, also a transcriptional target of hedgehog signaling in many cell types, was not increased. GLI1 protein and mRNA, and PTCH1 and sonic hedgehog (SHH) proteins were elevated in 3 of 10 breast cancers; however, PTCH1 transcripts were not consistently increased. Hedgehog-mediated transcription, as indicated by a reporter of GLI-dependent promoter activity and by expression of GLI1 transcripts, was reduced by the hedgehog pathway inhibitor cyclopamine in both MDA-MB-435 cancer epithelial cells and MCF10AT epithelial cells, a cell line derived from benign breast. However, cyclopamine reduced viability of cancer epithelial cell lines, including MDA-MB-435, but did not specifically affect fibroblasts or epithelial cells from benign breast, including MCF10AT. Treatment with sonic hedgehog ligand diminished the cyclopamine-induced reduction in GLI-dependent promoter activity in MCF10AT and MDA-MB-435 and viability of MDA-MB-435. These results demonstrate modulation of GLI-mediated transcription in both cancer and benign-derived epithelial cells by cyclopamine and sonic hedgehog, and further suggest that hedgehog signaling contributes to the survival of only the cancer epithelial cells. Determination as to whether the increase in GLI1 and SHH expression in breast cancer indicates a significant increase in hedgehog signaling will require further evaluation.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Mama/fisiología , Proteínas Hedgehog/fisiología , Alcaloides de Veratrum/farmacología , Secuencia de Bases , Mama/efectos de los fármacos , Línea Celular Tumoral , Cartilla de ADN , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiologíaRESUMEN
KLF4 is induced upon growth-arrest in vitro and during epithelial maturation in vivo, and is essential for proper cell fate specification of post-mitotic cells. In spite of a normal role in post-mitotic cells, expression is upregulated and constitutive in certain tumor types. KLF4 functions as an oncogene in vitro, and enforced expression in basal cells of mouse skin rapidly induces lesions similar to hyperplasia, dysplasia and squamous cell carcinoma (SCC). Here we used conditional expression to characterize early steps in KLF4-mediated tumor initiation. In contrast to SCC-like lesions that result when using a conditional, keratin 14 promoter-dependent strategy, lower conditional expression achieved using a MMTV promoter induced only epidermal cycling within morphologically normal skin, a process we termed occult cell turnover. Surprisingly, KLF4-induced hyperplastic lesions showed increased transgene-derived mRNA and protein in maturing, PCNA-negative cells, a property of endogenous KLF4. In contrast, hyperplastic lesions induced by GLI1, a control, showed uniform transgene expression. In KLF4-induced dysplasia and SCC the complementarity of KLF4 and PCNA was replaced by concordance of the two proteins. These studies show that KLF4 transcripts are normally suppressed in cycling cells in a promoter-independent fashion, consistent with a post-transcriptional control, and reveal loss of this control in the transition from hyperplasia to dysplasia. Like the mouse tumors, human cutaneous SCCs and adjacent dysplasias frequently showed maturation-independence of KLF4, with co-expression of KLF4 and PCNA. A smaller subset of human SCCs showed complementarity of KLF4 and PCNA, similar to hyperplastic mouse skin. The results identify parallels between a mouse model and human primary tumors, and show that successive increases of KLF4 in the nuclei of basal keratinocytes leads to occult cell turnover followed by hyperplasia, dysplasia, and invasive SCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Transformación Celular Neoplásica/inducido químicamente , Factores de Transcripción de Tipo Kruppel/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Neoplasias Cutáneas/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Doxorrubicina/farmacología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Transgénicos , Antígeno Nuclear de Célula en Proliferación/genética , ARN Mensajero/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transgenes , Proteína con Dedos de Zinc GLI1RESUMEN
PURPOSE: The Krüppel-like transcription factor KLF4/GKLF induces both malignant transformation and a slow-growth phenotype in vitro. Although KLF4 expression is increased in most cases of breast cancer, it was unknown whether these cases represent a distinct subtype with a different clinical outcome. EXPERIMENTAL DESIGN: We examined expression of KLF4 by immunostaining 146 cases of human primary infiltrating ductal carcinoma of the breast. Staining patterns were correlated with clinical outcome and with established prognostic factors. RESULTS: Subcellular localization exhibited case-to-case variation. Tumors with high nuclear staining and low cytoplasmic staining were termed type 1. For patients with early-stage disease (i.e., stage I or IIA), type 1 staining was associated with eventual death because of breast cancer (hazard ratio, 2.8; 95% confidence interval, 1.23-6.58; P = 0.011). The association was stronger in patients with early-stage cancer and small primary tumors (i.e., < or =2.0 cm in diameter; hazard ratio, 4.3; 95% confidence interval, 1.75-10.62; P < 0.001). For patients with early-stage disease, multivariate analysis indicated that type 1 staining was independently associated with outcome (adjusted hazard ratio 2.6; 95% confidence interval, 1.10-6.05; P = 0.029). Type 1 staining was also associated with high histological grade (P = 0.032), increased expression of Ki67 (P = 0.016), and reduced expression of BCL2 (P = 0.032). In vitro, KLF4 was localized within the nucleus of transformed RK3E epithelial cells, consistent with a nuclear function of this transcription factor during induction of malignant transformation. CONCLUSIONS: The results suggest that localization of KLF4 in the nucleus of breast cancer cells is a prognostic factor and identify KLF4 as a marker of an aggressive phenotype in early-stage infiltrating ductal carcinoma.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/biosíntesis , Factores de Transcripción/biosíntesis , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Transformación Celular Neoplásica , Citoplasma/metabolismo , Citosol/metabolismo , ADN Complementario/metabolismo , Supervivencia sin Enfermedad , Epítopos/química , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/biosíntesis , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Análisis Multivariante , Fenotipo , Plásmidos/metabolismo , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Análisis de Regresión , Factores de Tiempo , Transfección , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
(2E,4E,6Z,8E)-8-(3',4'-Dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid, 9cUAB30, is a selective rexinoid that displays substantial chemopreventive capacity with little toxicity. 4-Methyl-UAB30, an analogue of 9cUAB30, is a potent RXR agonist but caused increased lipid biosynthesis unlike 9cUAB30. To evaluate how methyl substitution influenced potency and lipid biosynthesis, we synthesized four 9cUAB30 homologues with methyl substitutions at the 5-, 6-, 7-, or 8-position of the tetralone ring. The syntheses and biological evaluations of these new analogues are reported here along with the X-ray crystal structures of each homologue bound to the ligand binding domain of hRXRα. We demonstrate that each homologue of 9cUAB30 is a more potent agonist, but only the 7-methyl-9cUAB30 caused severe hyperlipidemia in rats. On the basis of the X-ray crystal structures of these new rexinoids and bexarotene (Targretin) bound to hRXRα-LBD, we reveal that each rexinoid, which induced hyperlipidemia, had methyl groups that interacted with helix 7 residues of the LBD.
Asunto(s)
Anticarcinógenos/química , Ácidos Grasos Insaturados/química , Hiperlipidemias/inducido químicamente , Naftalenos/química , Receptor alfa X Retinoide/agonistas , Animales , Anticarcinógenos/farmacología , Anticarcinógenos/toxicidad , Apoptosis/efectos de los fármacos , Bexaroteno , Sitios de Unión , Línea Celular , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Cristalografía por Rayos X , Ácidos Grasos Insaturados/farmacología , Ácidos Grasos Insaturados/toxicidad , Femenino , Humanos , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/prevención & control , Modelos Moleculares , Estructura Molecular , Naftalenos/farmacología , Naftalenos/toxicidad , Ratas , Receptor alfa X Retinoide/genética , Estereoisomerismo , Relación Estructura-Actividad , Tetrahidronaftalenos/farmacología , Tetrahidronaftalenos/toxicidad , Activación TranscripcionalRESUMEN
The transcription factors Notch1 and KLF4 specify epithelial cell fates and confer stem cell properties. Suggesting a functional relationship, each gene can act to promote or suppress tumorigenesis in a context-dependent manner, and alteration of KLF4 or Notch pathway genes in mice gives rise to similar phenotypes. Activation of a conditional allele of KLF4 in RK3E epithelial cells rapidly induces expression of Notch1 mRNA and the active, intracellular form of Notch1. KLF4-induced transformation was suppressed by knockdown of endogenous Notch1 using siRNA or an inhibitor of gamma-secretase. Chromatin immunoprecipitation assay shows that KLF4 binds to the proximal Notch1 promoter in human mammary epithelial cells, and siRNA-mediated suppression of KLF4 in human mammary cancer cells results in reduced expression of Notch1. Furthermore, KLF4 and Notch1 expression are correlated in primary human breast tumors (N = 89; Pearson analysis, r > 0.5, p < 0.0001). Like KLF4, Notch1 was previously shown to induce transformation of rat cells immortalized with adenovirus E1A, similar to RK3E cells. We therefore compared the signaling requirements for Notch1- or KLF4-induced malignant transformation of RK3E. As expected, transformation by Notch1 was suppressed by dominant-negative CSL or MAML1, inhibitors of canonical Notch1 signaling. However, these inhibitors did not suppress transformation by KLF4. Therefore, while KLF4-induced transformation requires Notch1, canonical Notch1 signaling is not required, and Notch1 may signal through a distinct pathway in cells with increased KLF4 activity. These results suggest that KLF4 could contribute to breast tumor progression by activating synthesis of Notch1 and by promoting signaling through a non-canonical Notch1 pathway.
Asunto(s)
Transformación Celular Neoplásica/genética , Factores de Transcripción de Tipo Kruppel/genética , Receptor Notch1/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/patología , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Immunoblotting , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Receptor Notch1/biosíntesis , Receptor Notch1/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The transcription factor KLF4 acts in post-mitotic epithelial cells to promote differentiation and functions in a context-dependent fashion as an oncogene. In the skin KLF4 is co-expressed with the nuclear receptors RARgamma and RXRalpha, and formation of the skin permeability barrier is a shared function of these three proteins. We utilized a KLF4-transgenic mouse model of skin cancer in combination with cultured epithelial cells to examine functional interactions between KLF4 and retinoic acid receptors. In cultured cells, activation of a conditional, KLF4-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in rapid upregulation of transcripts for nuclear receptors including RARgamma and RXRalpha. We tested retinoids in epithelial cell transformation assays, including an RAR-selective agonist (all-trans RA), an RXR-selective agonist (9-cis UAB30, rexinoid), and a pan agonist (9-cis RA). Unlike for several other genes, transformation by KLF4 was inhibited by each retinoid, implicating distinct nuclear receptor heterodimers as modulators of KLF4 transforming activity. When RXRalpha expression was suppressed by RNAi in cultured cells, transformation was promoted and the inhibitory effect of 9-cis UAB30 was attenuated. Similarly as shown for other mouse models of skin cancer, rexinoid prevented skin tumor initiation resulting from induction of KLF4 in basal keratinocytes. Rexinoid permitted KLF4 expression and KLF4-induced cell cycling, but attenuated the KLF4-induced misexpression of cytokeratin 1 in basal cells. Neoplastic lesions including hyperplasia, dysplasia and squamous cell carcinoma-like lesions were prevented for up to 30 days. Taken together, the results identify retinoid receptors including RXRalpha as ligand-dependent inhibitors of KLF4-mediated transformation or tumorigenesis.