RESUMEN
Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods.
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Neoplasias de la Mama/inmunología , Modelos Animales de Enfermedad , Receptores ErbB/inmunología , Formaldehído/química , Microondas , Receptores de Estrógenos/inmunología , Receptores de Progesterona/inmunología , Algoritmos , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , RatonesRESUMEN
Breast cancers with a basal-like gene signature are primarily triple-negative, frequently metastatic, and carry a poor prognosis. Basal-like breast cancers are enriched for markers of breast cancer stem cells as well as markers of epithelial-mesenchymal transition (EMT). While EMT is generally thought to be important in the process of metastasis, in vivo evidence of EMT in human disease remains rare. Here we report a novel model of human triple-negative breast cancer, the DKAT cell line, which was isolated from an aggressive, treatment-resistant triple-negative breast cancer that demonstrated morphological and biochemical evidence suggestive of phenotypic plasticity in the patient. The DKAT cell line displays a basal-like phenotype in vitro when cultured in serum-free media, and undergoes phenotypic changes consistent with EMT/MET in response to serum-containing media, a unique property among the breast cancer cell lines we tested. This EMT is marked by increased expression of the transcription factor Zeb1, and Zeb1 is required for the enhanced migratory ability of DKAT cells in the mesenchymal state. DKAT cells also express progenitor-cell markers, and single DKAT cells are able to generate tumorspheres containing both epithelial and mesenchymal cell types. In vivo, as few as ten DKAT cells are capable of forming xenograft tumors which display a range of epithelial and mesenchymal phenotypes. The DKAT model provides a novel model to study the molecular mechanisms regulating phenotypic plasticity and the aggressive biology of triple-negative breast cancers.
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Neoplasias de la Mama/patología , Adulto , Animales , Médula Ósea/patología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Medio de Cultivo Libre de Suero/farmacología , Citogenética , Transición Epitelial-Mesenquimal , Exones , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Cariotipificación , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Fenotipo , PronósticoRESUMEN
Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex(3)HexNAc), 1485.530 (Hex(3)HexNAc(4)Fuc), 1809.639 (Hex(5)HexNAc(4)Fuc), and 1905.630 (Man(9)), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man(6-9)), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man(9), m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man(9) to produce complex and hybrid type oligosaccharides.
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Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Manosa/sangre , Polisacáridos/sangre , Animales , Conformación de Carbohidratos , Femenino , Humanos , Manosa/química , Ratones , Metástasis de la Neoplasia , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The ErbB2 and ErbB3 receptor tyrosine kinases act synergistically to promote cellular properties associated with tumor development. Previous studies indicate that endogenous ErbB3 protein is markedly elevated in mouse mammary tumors induced by transgenic ErbB2 overexpression. However, this occurs in the absence of elevated ErbB3 transcript, indicating that post-transcriptional regulatory mechanisms play crucial roles in suppressing ErbB3 protein in normal tissue. Our previous studies also demonstrate that protein levels of Nrdp1, an E3 ubiquitin ligase that targets ErbB3 for degradation, are markedly suppressed in tumors from ErbB2 transgenic animals relative to normal tissue. Here we demonstrate that transgenic expression of Nrdp1 cDNA in the mouse mammary gland is not sufficient to suppress elevated ErbB3 levels or tumor initiation and growth in ErbB2 transgenic mice. Unexpectedly, Nrdp1 protein is absent in tumors from Nrdp1/ErbB2 bigenic mice, and real time PCR analysis indicates that Nrdp1 protein levels are suppressed post-transcriptionally. Nrdp1 protein is more resistant to proteasome-dependent degradation when exogenously expressed in cultured MCF10A nontransformed human breast epithelial cells than in breast tumor cells. These observations indicate that mammary tumors use potent post-transcriptional mechanisms to suppress Nrdp1 protein levels and that protein destabilization may play a central role in Nrdp1 loss in tumors.
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Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Neoplasias Mamarias Animales/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Línea Celular , Femenino , Humanos , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/genética , Ratones , Ratones Transgénicos , Estabilidad Proteica , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Little is known about outcomes of glenoid labral repair in Major League Baseball (MLB) pitchers. We hypothesized that following repair, pitching performance would not be significantly different from an uninjured cohort. Fifty-one pitchers were identified who pitched in at least one MLB game prior to undergoing isolated glenoid labral repair. For the three years prior to and following surgery, demographic and performance variables were analyzed for an association with labral injury and repair, and compared to a control cohort of MLB pitchers without history of repair. Following surgery, 72.5% of pitchers returned to MLB at a mean of 13.1 months with no significant change in performance. Starting pitchers had a higher risk of labral injury requiring repair (p< or =0.05). Pitchers that returned to play averaged more innings pitched in the seasons prior to surgery and had a higher body mass index than those that did not return to play (p< or =0.05). Approximately 70% of MLB pitchers undergoing labral repair can be expected to return to competition postoperatively with no significant change in performance. Starting pitchers are more likely to undergo repair, but pitchers with greater preoperative innings pitched per season have a greater likelihood of returning to play.
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Traumatismos del Brazo/cirugía , Béisbol/lesiones , Procedimientos Ortopédicos , Lesiones del Hombro , Articulación del Hombro/cirugía , Adulto , Traumatismos del Brazo/rehabilitación , Índice de Masa Corporal , Estudios de Casos y Controles , Intervalos de Confianza , Humanos , Modelos Logísticos , Masculino , Análisis Multivariante , Factores de Riesgo , Resultado del TratamientoRESUMEN
The role of the PEA3 subfamily of Ets transcription factors in breast neoplasia is controversial. Although overexpression of PEA3 (E1AF/ETV4), and of the related factors ERM (ETV5) and ER81 (ETV1), have been observed in human and mouse breast tumors, PEA3 factors have also been ascribed a tumor suppressor function. Here, we utilized the MMTV/Wnt1 mouse strain to further interrogate the role of PEA3 transcription factors in mammary tumorigenesis based on our previous observation that Pea3 is highly expressed in MMTV/Wnt1 mammary tumors. Pea3 expression in mouse mammary tissues was visualized using a Pea3(NLSlacZ) reporter strain. In normal mammary glands, Pea3 expression is predominantly confined to myoepithelial cells. Wnt1 transgene expression induced marked amplification of this cell compartment in nontumorous mammary glands, accompanied by an apparent increase in Pea3 expression. The pattern of Pea3 expression in MMTV/Wnt1 mammary glands recapitulated the cellular profile of activated beta-catenin/TCF signaling, which was visualized using both beta-catenin immunohistochemistry and the beta-catenin/TCF-responsive reporter Axin2(NLSlacZ). To test the requirement for PEA3 factors in Wnt1-induced tumorigenesis, we employed a mammary-targeted dominant negative PEA3 transgene, DeltaNPEA3En. Expression of DeltaNPEA3En delayed early-onset tumor formation in MMTV/Wnt1 virgin females (P = 0.03), suggesting a requirement for PEA3 factor function for Wnt1-driven tumor formation. Consistent with this observation, expression of the DeltaNPEA3En transgene was profoundly reduced in mammary tumors compared to nontumorous mammary glands from bigenic MMTV/Wnt1, MMTV/DeltaNPEA3En mice (P = 0.01). Our data provide the first description of Wnt1-mediated expansion of the Pea3-expressing myoepithelial compartment in nontumorous mammary glands. Consistent with this observation, mammary myoepithelium was selectively responsive to Wnt1. Together these data suggest the MMTV/Wnt1 strain as a potential model of basal breast cancer. Furthermore, this study provides evidence for a protumorigenic role of PEA3 factors in breast neoplasia, and supports targeting the PEA3 transcription factor family in breast cancer.
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Neoplasias Mamarias Experimentales/fisiopatología , Factores de Transcripción/fisiología , Proteína Wnt1/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Femenino , Inmunohistoquímica , Neoplasias Mamarias Experimentales/genética , Virus del Tumor Mamario del Ratón , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Transcripción TCF/metabolismo , Factores de Transcripción/genética , Transgenes , Proteína Wnt1/genética , beta Catenina/metabolismoRESUMEN
To provide a continuous and prolonged delivery of the substrate D-luciferin for bioluminescence imaging in vivo, luciferin was encapsulated into liposomes using either the pH gradient or acetate gradient method. Under optimum loading conditions, 0.17 mg luciferin was loaded per mg of lipid with 90-95% encapsulation efficiency, where active loading was 6 to 18-fold higher than that obtained with passive loading. Liposomal luciferin in a long-circulating formulation had good shelf stability, with 10% release over 3-month storage at 4 degrees C. Pharmacokinetic profiles of free and liposomal luciferin were then evaluated in transgenic mice expressing luciferase. In contrast to rapid in vivo clearance of free luciferin (t(1/2)=3.54 min), luciferin encapsulated into long-circulating liposomes showed a prolonged release over 24h. The first-order release rate constant of luciferin from long-circulating liposomes, as estimated from the best fit of the analytical model to the experimental data, was 0.01 h(-1). Insonation of luciferin-loaded temperature-sensitive liposomes directly injected into one tumor of Met1-luc tumor-bearing mice resulted in immediate emission of light. Systemic injection of luciferin-loaded long-circulating liposomes into Met1-luc tumor-bearing mice, followed by unilateral ultrasound-induced hyperthermia, produced a gradual increase in radiance over time, reaching a peak at 4-7 h post-ultrasound.
Asunto(s)
Benzotiazoles/administración & dosificación , Sistemas de Liberación de Medicamentos , Luminiscencia , Sustancias Luminiscentes/administración & dosificación , Neoplasias Mamarias Experimentales/patología , Animales , Benzotiazoles/química , Benzotiazoles/farmacocinética , Línea Celular Tumoral , Química Farmacéutica , Preparaciones de Acción Retardada , Composición de Medicamentos , Estabilidad de Medicamentos , Femenino , Concentración de Iones de Hidrógeno , Hipertermia Inducida , Inyecciones Intralesiones , Inyecciones Intravenosas , Liposomas , Luciferasas/genética , Luciferasas/metabolismo , Sustancias Luminiscentes/química , Sustancias Luminiscentes/farmacocinética , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Transgénicos , Modelos Biológicos , Permeabilidad , Solubilidad , Temperatura , Transfección , UltrasonidoRESUMEN
The ErbB2 receptor tyrosine kinase is overexpressed in approximately 25% of breast tumors and contributes to poor patient prognosis and therapeutic resistance. Here, we examine the role of the recently discovered ErbB negative regulator LRIG1 in ErbB2(+) breast cancer. We observe that LRIG1 protein levels are significantly suppressed in ErbB2-induced mammary tumors in transgenic mice as well as in the majority of ErbB2(+) human breast tumors. These observations raise the possibility that LRIG1 loss could contribute to the initiation or growth of ErbB2(+) breast tumors. RNA interference-mediated knockdown of endogenous LRIG1 in the ErbB2-overexpressing breast tumor cell lines MDA-MB-453 and BT474 further elevates ErbB2 in these cells and augments cellular proliferation. In contrast, ectopic expression of LRIG1 reverses these trends. Interestingly, we observe that LRIG1 protein levels are suppressed in response to ErbB receptor activation in breast tumor cells but are unaffected by ErbB activation in immortalized nontransformed breast epithelial cells. Our observations indicate that the suppression of LRIG1 protein levels is a common feature of breast tumors. Moreover, our observations point to the existence of a feed-forward regulatory loop in breast tumor cells where aberrant ErbB2 signaling suppresses LRIG1 protein levels, which in turn contributes to ErbB2 overexpression.
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Neoplasias de la Mama/etiología , Glicoproteínas de Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptor ErbB-2/fisiología , Animales , Neoplasias de la Mama/química , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Genes erbB-2 , Humanos , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/antagonistas & inhibidores , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/análisis , Receptor ErbB-2/análisis , Transducción de SeñalAsunto(s)
Metilación de ADN , Ácido Fólico , Neoplasias Mamarias Experimentales/genética , Animales , Carcinoma Intraductal no Infiltrante/patología , Dieta , Femenino , Ácido Fólico/administración & dosificación , Ácido Fólico/efectos adversos , Ácido Fólico/metabolismo , Expresión Génica , Genes erbB-2/genética , Humanos , RatonesRESUMEN
The mouse model for breast cancer has developed into a most effective means of dissecting and understanding this devastating disease. The inbred transgenic mouse lends itself to biological, molecular, immunological, and genetic studies. The observation, dissection, transplantation, and subsequent amplification of precancerous mammary lesions and tumors give the scientist the means to readily study the tissues and design interventions and therapeutic drugs for the future eventual control of breast cancer. There are many inbred strains of mice, selected for specific characteristics. The mouse is easy to handle, breeds well, and does not require extensive facilities, funding, and handling such as monkeys, chimps, and other animal models. A huge advantage is the capability for the transplantation of tissues as well as gene manipulation, which make the transgenic mouse a major research resource. The mouse has served the scientific community well for over a century and will continue to do so in the quest for understanding breast cancer and other diseases.
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Neoplasias de la Mama/genética , Modelos Animales de Enfermedad , Neoplasias Mamarias Animales/genética , Animales , Regulación Neoplásica de la Expresión Génica , Humanos , Oncología Médica/métodos , Ratones , Ratones Transgénicos , Modelos Biológicos , Trasplante de Neoplasias , OncogenesRESUMEN
INTRODUCTION: The 'MINO' (mammary intraepithelial neoplasia outgrowth) mouse model of ductal carcinoma in situ (DCIS) consists of six lines with distinct morphologic phenotypes and behavior, each meeting experimentally defined criteria for 'precancer'. Specifically, these lines grow orthotopically in cleared mammary fat pads and consistently progress to an invasive phenotype that is capable of ectopic growth. Transition to carcinoma has a consistent latency for each line, and three of the lines also exhibit pulmonary metastatic potential. METHODS: Gland cleared orthotopic transplanted precancer MINO tissues were analyzed by bacterial artifical chromosome and oligo array comparative genomic hybridization, microsatellite PCR, and telomerase repeat amplification assay. MINO cells were dissociated and cultured in three dimensional culture and transplanted in syngeneic gland cleared mammary fat pads. RESULTS: Comparative genomic hybridization shows that the precancer and invasive tumors are genetically stable, with low level changes including whole chromosome gains in some lines. No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally. Microsatellite analysis shows a low frequency of alterations that are predominantly permanent within a MINO line. Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland. Dissociation of the precancer lesion cells and three dimensional 'spheroid' culture of single cells reveals a bipotential for myoepithelial and luminal differentiation and the formation of unique three-dimensional 'MINOspheres'. These MINOspheres exhibit features that are intermediate between spheroids that are derived from normal and carcinoma cells. Transplantation of a single cell derived MINOsphere recapitulates the outgrowth of the precancer morphology and progression to carcinoma. CONCLUSION: These data establish a precancer 'stem' cell that is capable of self-renewal and multilineage differentiation as the origin of invasive cancer. Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.
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Neoplasias Mamarias Animales/metabolismo , Células Madre Neoplásicas/citología , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/patología , Neoplasias Mamarias Animales/patología , Ratones , Repeticiones de Microsatélite , Metástasis de la Neoplasia , Trasplante de Neoplasias , Células Madre Neoplásicas/patología , Hibridación de Ácido Nucleico , Fenotipo , Reacción en Cadena de la Polimerasa , Lesiones Precancerosas , Telomerasa/metabolismoRESUMEN
T-cadherin delineates endothelial, myoepithelial, and ductal epithelial cells in the normal mouse mammary gland, and becomes progressively restricted to the vasculature during mammary tumorigenesis. To test the function of T-cadherin in breast cancer, we inactivated the T-cadherin (Cdh13) gene in mice and evaluated tumor development and pathology after crossing the mutation into the mouse mammary tumor virus (MMTV)-polyoma virus middle T (PyV-mT) transgenic model. We report that T-cadherin deficiency limits mammary tumor vascularization and reduces tumor growth. Tumor transplantation experiments confirm the stromal role of T-cadherin in tumorigenesis. In comparison with wild-type MMTV-PyV-mT controls, T-cadherin-deficient tumors are pathologically advanced and metastasize to the lungs. T-cadherin is a suggested binding partner for high molecular weight forms of the circulating, fat-secreted hormone adiponectin. We discern adiponectin in association with the T-cadherin-positive vasculature in the normal and malignant mammary glands and report that this interaction is lost in the T-cadherin null condition. This work establishes a role for T-cadherin in promoting tumor angiogenesis and raises the possibility that vascular T-cadherin-adiponectin association may contribute to the molecular cross-talk between tumor cells and the stromal compartment in breast cancer.
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Adiponectina/biosíntesis , Cadherinas/fisiología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/metabolismo , Neovascularización Patológica , Adiponectina/metabolismo , Animales , Cadherinas/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesisRESUMEN
BACKGROUND: Human breast cancer is a heterogeneous disease, histopathologically, molecularly and phenotypically. The molecular basis of this heterogeneity is not well understood. We have used a mouse model of DCIS that consists of unique lines of mammary intraepithelial neoplasia (MIN) outgrowths, the premalignant lesion in the mouse that progress to invasive carcinoma, to understand the molecular changes that are characteristic to certain phenotypes. Each MIN-O line has distinguishable morphologies, metastatic potentials and estrogen dependencies. METHODS: We utilized oligonucleotide expression arrays and high resolution array comparative genomic hybridization (aCGH) to investigate whole genome expression patterns and whole genome aberrations in both the MIN-O and tumor from four different MIN-O lines that each have different phenotypes. From the whole genome analysis at 35 kb resolution, we found that chromosome 1, 2, 10, and 11 were frequently associated with whole chromosome gains in the MIN-Os. In particular, two MIN-O lines had the majority of the chromosome gains. Although we did not find any whole chromosome loss, we identified 3 recurring chromosome losses (2F1-2, 3E4, 17E2) and two chromosome copy number gains on chromosome 11. These interstitial deletions and duplications were verified with a custom made array designed to interrogate the specific regions at approximately 550 bp resolution. RESULTS: We demonstrated that expression and genomic changes are present in the early premalignant lesions and that these molecular profiles can be correlated to phenotype (metastasis and estrogen responsiveness). We also identified expression changes associated with genomic instability. Progression to invasive carcinoma was associated with few additional changes in gene expression and genomic organization. Therefore, in the MIN-O mice, early premalignant lesions have the major molecular and genetic changes required and these changes have important phenotypic significance. In contrast, the changes that occur in the transition to invasive carcinoma are subtle, with few consistent changes and no association with phenotype. CONCLUSION: We propose that the early lesions carry the important genetic changes that reflect the major phenotypic information, while additional genetic changes that accumulate in the invasive carcinoma are less associated with the overall phenotype.
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Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Heterogeneidad Genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Animales , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Línea Celular Tumoral , Inestabilidad Cromosómica , Aberraciones Cromosómicas , Cromosomas de los Mamíferos , Femenino , Regulación Neoplásica de la Expresión Génica , Hormonas Gonadales/farmacología , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Metástasis de la Neoplasia/patología , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patologíaRESUMEN
Dysregulation of ErbB receptor tyrosine kinases is thought to promote mammary tumor progression by stimulating tumor cell growth and invasion. Overexpression and aberrant activation of ErbB2/HER2 confer aggressive and malignant characteristics to breast cancer cells, and patients displaying ErbB2-amplified breast cancer face a worsened prognosis. Recent studies have established that ErbB2 and ErbB3 are commonly co-overexpressed in breast tumor cell lines and in patient samples. ErbB2 heterodimerizes with and activates the ErbB3 receptor, and the two receptors synergize in promoting growth factor-induced cell proliferation, transformation, and invasiveness. Our previous studies have shown that the neuregulin receptor degradation protein-1 (Nrdp1) E3 ubiquitin ligase specifically suppresses cellular ErbB3 levels by marking the receptor for proteolytic degradation. Here, we show that overexpression of Nrdp1 in human breast cancer cells results in the suppression of ErbB3 levels, accompanied by the inhibition of cell growth and motility and the attenuation of signal transduction pathways. In contrast, either Nrdp1 knockdown or the overexpression of a dominant-negative form enhances ErbB3 levels and cellular proliferation. Additionally, Nrdp1 expression levels inversely correlate with ErbB3 levels in primary human breast cancer tissue and in a mouse model of ErbB2 mammary tumorigenesis. Our observations suggest that Nrdp1-mediated ErbB3 degradation suppresses cellular growth and motility, and that Nrdp1 loss in breast tumors may promote tumor progression by augmenting ErbB2/ErbB3 signaling.
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Neoplasias de la Mama/patología , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ecdisterona/análogos & derivados , Ecdisterona/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Vectores Genéticos/genética , Humanos , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/farmacología , Neurregulina-1 , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Interferente Pequeño/genética , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Retroviridae/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transfección , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
PURPOSE: Rapamycin has been shown to have antitumor effects in various tumor models. To study the effect of rapamycin at different stages of breast cancer development, we used two unique mouse models of breast cancer with activated phosphatidylinositol 3-kinase (PI3K) pathway. Met-1 tumors are highly invasive and metastatic, and mammary intraepithelial neoplasia-outgrowths (MIN-O), a model for human ductal carcinoma in situ, are transplantable premalignant mammary lesions that develop invasive carcinoma with predictable latencies. Both of these models were derived from mammary lesions in Tg(MMTV-PyV-mT) mice. EXPERIMENTAL DESIGN: Met-1 tumors were used to study the effect of rapamycin treatment on invasive disease. Transplanted MIN-O model was used to study the effect of rapamycin on premalignant mammary lesions. Animals were in vivo micro-positron emission tomography imaged to follow the lesion growth and transformation to tumor during the treatment. Cell proliferation, angiogenesis, and apoptosis was assayed by immunohistochemistry. RESULTS: Rapamycin inhibited in vitro tumor cell proliferation and in vivo Met-1 tumor growth. The growth inhibition was correlated with dephosphorylation of mammalian target of rapamycin (mTOR) targets. Rapamycin treatment significantly reduced the growth of the premalignant MIN-O lesion, as well as tumor incidence and tumor burden. Growth inhibition was associated with reduced cell proliferation and angiogenesis and increased apoptosis. CONCLUSIONS: In PyV-mT mouse mammary models, rapamycin inhibits the growth of premalignant lesions and invasive tumors. Although the inhibitory effect of rapamycin was striking, rapamycin treatment did not completely obliterate the lesions.
Asunto(s)
Carcinoma Intraductal no Infiltrante/prevención & control , Proliferación Celular/efectos de los fármacos , Neoplasias Mamarias Experimentales/prevención & control , Lesiones Precancerosas/prevención & control , Sirolimus/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/fisiología , Apoptosis/efectos de los fármacos , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Tomografía de Emisión de Positrones , Lesiones Precancerosas/irrigación sanguínea , Lesiones Precancerosas/patología , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR , Factores de TiempoRESUMEN
INTRODUCTION: Ductal carcinoma in situ (DCIS) is a noninvasive premalignant lesion and is considered a precursor to invasive carcinoma. DCIS accounts for nearly 20% of newly diagnosed breast cancer, but the lack of experimentally amenable in vivo DCIS models hinders the development of treatment strategies. Here, we demonstrate the utility of a mouse transplantation model of DCIS for chemoprevention studies using selective estrogen receptor modulators (SERMs). This model consists of a set of serially transplanted lines of genetically engineered mouse mammary intraepithelial neoplasia (MIN) outgrowth (MIN-O) tissue that have stable characteristics. We studied the ovarian-hormone-responsiveness of one of the lines with a particular focus on the effects of two related SERMs, tamoxifen and ospemifene. METHODS: The estrogen receptor (ER) status and ovarian-hormone-dependence of the mouse MIN outgrowth tissue were determined by immunohistochemistry and ovarian ablation. The effects of tamoxifen and ospemifene on the growth and tumorigenesis of MIN outgrowth were assessed at 3 and 10 weeks after transplantation. The effects on ER status, cell proliferation, and apoptosis were studied with immunohistochemistry. RESULTS: The MIN-O was ER-positive and ovarian ablation resulted in reduced MIN-O growth and tumor development. Likewise, tamoxifen and ospemifene treatments decreased the MIN growth and tumor incidence in comparison with the control (P < 0.01). Both SERMs significantly decreased cell proliferation. Between the two SERM treatment groups, there were no statistically significant differences in MIN-O size, tumor latency, or proliferation rate. In contrast, the ospemifene treatment significantly increased ER levels while tamoxifen significantly decreased them. CONCLUSION: Tamoxifen and ospemifene inhibit the growth of premalignant mammary lesions and the progression to invasive carcinoma in a transplantable mouse model of DCIS. The inhibitory effects of these two SERMs are similar except for their effects on ER modulation. These differences in ER modulation may suggest different mechanisms of action between the two related SERMs and may portend different long-term outcomes. These data demonstrate the value of this model system for preclinical testing of antiestrogen or other therapies designed to prevent or delay the malignant transformation of premalignant mammary lesions in chemoprevention.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Carcinoma Intraductal no Infiltrante/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Animales , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/patología , Quimioprevención , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Mamarias Experimentales/patología , Ratones , Invasividad Neoplásica , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/fisiologíaRESUMEN
The inducible prostaglandin synthase cyclooxygenase-2 (Cox-2) is overexpressed in approximately 40% of human breast cancers and at higher frequencies in preinvasive ductal carcinoma in situ (DCIS). Cox-2 expression is particularly associated with overexpression of human epidermal growth factor receptor 2 (HER2/neu). To definitively interrogate the role of Cox-2 in mammary neoplasia, we have used a genetic approach, crossing Cox-2-deficient mice with a HER2/neu transgenic strain, MMTV/NDL. At 20 weeks of age, mammary glands from virgin MMTV/NDL females contained multiple focal tumors, or mammary intraepithelial neoplasias, which histologically resembled human DCIS. Mammary tumor multiplicity and prostaglandin E2 (PGE2) levels were significantly decreased in Cox-2 heterozygous and knockout animals relative to Cox-2 wild-type controls. Notably, the proportion of larger tumors was decreased in Cox-2-deficient mice. HER2/neu-induced mammary hyperplasia was also substantially reduced in Cox-2 null mice. Additionally, mammary glands from Cox-2 knockout mice exhibited a striking reduction in vascularization, and expression of proangiogenic genes was correspondingly reduced. Decreased vascularization was observed both in dysplastic and normal-appearing regions of Cox-2-null mammary glands. Our data provide the first genetic evidence that Cox-2 contributes to HER2/neu-induced mammary tumorigenesis. This finding may help to explain the reduced risk of breast cancer associated with regular use of nonsteroidal anti-inflammatory drugs.
Asunto(s)
Ciclooxigenasa 2/deficiencia , Neoplasias Mamarias Experimentales/genética , Receptor ErbB-2/genética , Animales , Ciclooxigenasa 2/genética , Femenino , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/enzimología , Neovascularización Patológica/genéticaRESUMEN
Two cell lines, Met-1(fvb2) and DB-7(fvb2), with different metastatic potential, were derived from mammary carcinomas in FVB/N-Tg(MMTV-PyVmT) and FVB/N-Tg(MMTV-PyVmT ( Y315F/Y322F )) mice, transplanted into syngeneic FVB/N hosts and characterized. The lines maintain a stable morphological and biological phenotype after multiple rounds of in vitro culture and in vivo transplantation. The Met-1(fvb2) line derived from a FVB/N-Tg(MMTV-PyVmT) tumor exhibits invasive growth and 100% metastases when transplanted into the females FVB/N mammary fat pad. The DB-7(fvb2) line derived from the FVB/N-Tg(MMTV-PyVmT ( Y315F/Y322F )) with a "double base" modification at Y315F/Y322F exhibits more rapid growth when transplanted into the mammary fat pad, but a lower rate of metastasis (17%). The Met1(fvb2) cells show high activation of AKT, while DB-7(fvb2) cells show very low levels of AKT activation. The DNA content and gene expression levels of both cell lines are stable over multiple generations. Therefore, these two cell lines provide a stable, reproducible resource for the study of metastasis modulators, AKT molecular pathway interactions, and gene target and marker discovery.
Asunto(s)
Carcinoma/patología , Línea Celular Tumoral , Neoplasias Mamarias Experimentales/patología , Animales , Carcinoma/genética , Carcinoma/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Metástasis de la Neoplasia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Estrógenos/análisis , Receptores de Estrógenos/metabolismoRESUMEN
It has been speculated that weekly paclitaxel enhances antiangiogenesis and, hence, results in a greater inhibition of cancer growth than the 3-week schedule. We compared the weekly and 3-week schedules of paclitaxel in inhibiting angiogenesis, tumor growth and bone marrow hematopoiesis in a lung cancer model. Vehicle or paclitaxel was administered i.p. to three groups of nude mice bearing a human lung cancer. The vehicle was given weekly for six doses or every 3 weeks for two doses (Group A). Paclitaxel was administered at 20 mg/kg/week for six doses (Group B) or 60 mg/kg/3 weeks for two doses (Group C). The tumor growth rate was reduced by 50% equally in both the paclitaxel-treated groups. Intratumoral microvasculature was reduced by 70% in each paclitaxel-treated group. However, white blood cell count was significantly reduced in Group C in comparison with that of Group A or B. We conclude that in this model, angiogenesis and tumor growth were inhibited to the same extent when paclitaxel was administered on a weekly or 3-week schedule. Inhibition of tumor growth by paclitaxel was associated with suppression of angiogenesis. Weekly administration of paclitaxel resulted in a lower degree of leukopenia than with the 3-week schedule, mimicking the clinical setting.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de Crecimiento/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Paclitaxel/administración & dosificación , Animales , Línea Celular Tumoral , Esquema de Medicación , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The development of models to investigate the pathobiology of premalignant breast lesions is a critical prerequisite for development of breast cancer prevention and early intervention strategies. Using tissue transplantation techniques, we modified the widely used polyomavirus middle T (PyV-mT) transgenic mouse model of breast cancer to study the premalignant stages of tumorigenesis. Premalignant atypical lesions were isolated from PyV-mT transgenic mice and used to generate two sets of three mammary intraepithelial neoplasia (MIN) outgrowth lines. Investigation of these six unique lines, each of which fulfills the criteria for MIN, has provided new information regarding the biology of PyV-mT-induced neoplasia. Although expression of the PyV-mT transgene was the primary initiating event for all lines, they exhibited different tumor latencies, metastatic potentials, and morphologies. Six distinguishable morphologic patterns of differentiation were identified within the premalignant outgrowths that are likely to represent several tumorigenic pathways. Further, several tumor phenotypes developed from each line and the tumors developing from the six lines had different metastatic potentials. These observations are consistent with the hypothesis that distinct pathways of PyV-mT-initiated neoplastic progression lead to different outcomes with respect to latency and metastasis. The MIN outgrowth lines share several characteristics with precursors of human breast cancer including the observation that gene expression profiles of tumors are more similar to those of the MIN outgrowth line outgrowth from which they developed than to other tumors. These lines provide an opportunity to study the full range of events occurring secondary to PyV-mT expression in the mammary gland.