RESUMEN
Identification of conserved co-expression networks is a useful tool for clustering groups of genes enriched for common molecular or cellular functions [1]. The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2]. Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer. Here, we validate one of the highly connected genes as a metastasis associated gene. Tpx2, the most highly connected gene within a proliferation network specifically prognostic for estrogen receptor positive (ER+) breast cancers, enhances metastatic disease, but in a tumor autonomous, proliferation-independent manner. Histologic analysis suggests instead that variation of TPX2 levels within disseminated tumor cells may influence the transition between dormant to actively proliferating cells in the secondary site. These results support the co-expression network approach for identification of new metastasis-associated genes to provide new information regarding the etiology of breast cancer progression and metastatic disease.
Asunto(s)
Neoplasias de la Mama/genética , Redes Reguladoras de Genes , Neoplasias Pulmonares/genética , Animales , Apoptosis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Trasplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pronóstico , Transcriptoma , Carga TumoralRESUMEN
Recent advances in genome wide transcriptional analysis have provided greater insights into the etiology and heterogeneity of breast cancer. Molecular signatures have been developed that stratify the conventional estrogen receptor positive or negative categories into subtypes that are associated with differing clinical outcomes. It is thought that the expression patterns of the molecular subtypes primarily reflect cell-of-origin or tumor driver mutations. In this study however, using a genetically engineered mouse mammary tumor model we demonstrate that the PAM50 subtype signature of tumors driven by a common oncogenic event can be significantly influenced by the genetic background on which the tumor arises. These results have important implications for interpretation of "snapshot" expression profiles, as well as suggesting that incorporation of genetic background effects may allow investigation into phenotypes not initially anticipated in individual mouse models of cancer.
Asunto(s)
Neoplasias Pulmonares/genética , Neoplasias Mamarias Experimentales/genética , Transcriptoma , Animales , Animales no Consanguíneos , Femenino , Estudios de Asociación Genética , Sitios Genéticos , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Queratinas/metabolismo , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Masculino , Neoplasias Mamarias Experimentales/clasificación , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones TransgénicosRESUMEN
Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.
Asunto(s)
Fraccionamiento Químico/métodos , Cromatografía por Intercambio Iónico/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Complejos Multiproteicos/análisis , Cationes , Línea Celular Tumoral , Cromatografía Liquida , Factor de Transcripción GATA3/análisis , Factor de Transcripción GATA3/genética , Células HEK293 , Humanos , Espectrometría de Masas , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/análisis , Proteínas Represoras/genética , Reproducibilidad de los Resultados , TransfecciónRESUMEN
The development of metastatic cancer is associated with overexpression or downregulation of specific genes and cell regulatory pathways. Some of these genes and pathways may be involved in invasion and dissemination of tumor cells, while others may promote seeding, survival or growth of cells at specific distant sites. In this investigation, gene expression profiles of nonmetastasizing tumors generated by injecting mouse pheochromocytoma cells (MPCs) subcutaneously were compared to those of liver tumors generated by injecting the cells intravenously. Both were compared to the cultured parental cell line. Tumors in the liver have a route of spread, anatomical distribution, and growth environment similar to naturally metastasizing pheochromocytomas, while intravenous injection of cells bypasses the initial steps of metastasis occurring spontaneously from a primary tumor. Eight genes were upregulated in liver tumors, 15 in subcutaneous tumors and seven in both compared to the cultured cells. Using quantitative real-time PCR, expression of five genes (Metap2, Reck, S100a4, Timp2, and Timp3) was verified as significantly lower in liver tumors than in subcutaneous tumors. Downregulation of these genes has been previously been associated with malignancy of pheochromocytomas. These findings indicate that different microenvironments can differentially affect the expression of metastasis-related genes in pheochromocytomas, and that overexpression or underexpression of these genes need not be present when the tumor cells are initially disseminated. The hepatic localization of tumors formed by intravenously injected MPC cells and the tumors' gene expression profile resembling that of naturally occurring pheochromocytoma metastases support the use of this model to study pheochromocytoma metastasis.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Perfilación de la Expresión Génica , Neoplasias Hepáticas Experimentales/secundario , Feocromocitoma/genética , Feocromocitoma/secundario , Tejido Subcutáneo/patología , Neoplasias de las Glándulas Suprarrenales/patología , Neoplasias de las Glándulas Suprarrenales/secundario , Animales , Línea Celular Tumoral , Femenino , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , Feocromocitoma/patologíaRESUMEN
The past decade has witnessed the unveiling of a powerful new generation of genetically engineered mouse (GEM) models of human cancer, which are proving to be highly effective for elucidating cancer mechanisms and interrogating novel experimental therapeutics. This new generation of GEM models are well suited for chemoprevention research, particularly for investigating progressive stages of carcinogenesis, identifying biomarkers for early detection and intervention, and preclinical assessment of novel agents or combinations of agents. Here we discuss opportunities and challenges for the application of GEM models in prevention research, as well as strategies to maximize their relevance for human cancer.
Asunto(s)
Quimioprevención/métodos , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ratones Transgénicos , Neoplasias/prevención & control , Animales , Ensayos de Selección de Medicamentos Antitumorales/tendencias , Eficiencia , Genes Supresores de Tumor , Humanos , Ratones , Modelos Biológicos , Neoplasias/genéticaRESUMEN
The goal of this study was to develop a sensitive, simple, and widely applicable assay to measure copy numbers of specific mRNAs using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and identify a profile of gene expression closely associated with angiogenesis. We measured a panel of nine potential angiogenesis markers from a mouse transgenic model of prostate adenocarcinoma (TRAMP) and a mouse skin model of vascular endothelial growth factor (VEGF)-driven angiogenesis. In both models, expression of VEGF correlated with expression of mRNAs encoding other angiogenic cytokines (angiopoietin-1 and angiopoietin-2), endothelial cell receptor tyrosine kinases (Flt-1, KDR, Tie-1), and endothelial cell adhesion molecules (VE-cadherin, PECAM-1). Relative to control, in dermis highly stimulated by VEGF, the Ang-2 mRNA transcript numbers increased 35-fold, PECAM-1 and VE-cadherin increased 10-fold, Tie-1 increased 8-fold, KDR and Flt-1 each increased 4-fold, and Ang-1 increased 2-fold. All transcript numbers were correspondingly reduced in skin with less VEGF expression, indicating a relationship of each of these seven markers with VEGF. Thus, this study identifies a highly efficient method for precise quantification of a panel of seven specific mRNAs that correlate with VEGF expression and VEGF-induced neovascularization, and it provides evidence that real-time quantitative RT-PCR offers a highly sensitive strategy for monitoring angiogenesis.