RESUMO
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.
Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Progressão da Doença , Manose/sangue , Polissacarídeos/sangue , Animais , Configuração de Carboidratos , Feminino , Humanos , Manose/química , Camundongos , Metástase Neoplásica , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Mamárias Animais/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Linhagem Celular , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Transgênicos , Estabilidade Proteica , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
Assuntos
Traumatismos do Braço/cirurgia , Beisebol/lesões , Procedimentos Ortopédicos , Lesões do Ombro , Articulação do Ombro/cirurgia , Adulto , Traumatismos do Braço/reabilitação , Índice de Massa Corporal , Estudos de Casos e Controles , Intervalos de Confiança , Humanos , Modelos Logísticos , Masculino , Análise Multivariada , Fatores de Risco , Resultado do TratamentoRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Modelos Animais de Doenças , Neoplasias Mamárias Animais/genética , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Oncologia/métodos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Transplante de Neoplasias , OncogenesRESUMO
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.
Assuntos
Neoplasias Mamárias Animais/metabolismo , Células-Tronco Neoplásicas/citologia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Neoplasias Mamárias Animais/patologia , Camundongos , Repetições de Microssatélites , Metástase Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Hibridização de Ácido Nucleico , Fenótipo , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas , Telomerase/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/patologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Vetores Genéticos/genética , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/farmacologia , Neuregulina-1 , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/genética , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Retroviridae/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
In 3T3-L1 adipocytes, both insulin and endothelin 1 stimulate glucose transport via translocation of the GLUT4 glucose carrier from an intracellular compartment to the cell surface. Yet it remains uncertain as to whether both hormones utilize identical pathways and to what extent each depends on the heterotrimeric G protein Galphaq as an intermediary signaling molecule. In this study, we used a novel inducible system to rapidly and synchronously activate expression of a dominant inhibitory form of ADP-ribosylation factor 6, ARF6(T27N), in 3T3-L1 adipocytes and assessed its effects on insulin- and endothelin-stimulated hexose uptake. Expression of ARF6(T27N) in 3T3-L1 adipocytes was without effect on the ability of insulin to stimulate either 2-deoxyglucose uptake or the translocation of GLUT4 or GLUT1 to the plasma membrane. However, the same ARF6 inhibitory mutant blocked the stimulation of hexose uptake and GLUT4 translocation in response to either endothelin 1 or an activated form of Galphaq, Galphaq(Q209L). These results suggest that endothelin stimulates glucose transport through a pathway that is distinct from that utilized by insulin but is likely to depend on both a heterotrimeric G protein from the Gq family and the small G protein ARF6. These data are consistent with the interpretation that endothelin and insulin stimulate functionally different pools of glucose transporters to be redistributed to the plasma membrane.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/fisiologia , Adipócitos/metabolismo , Endotelina-1/metabolismo , Glucose/farmacocinética , Insulina/metabolismo , Proteínas Musculares , Células 3T3 , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Adenoviridae/genética , Animais , Northern Blotting , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endotelinas/metabolismo , Ativação Enzimática , Genes Dominantes , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Camundongos , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Plasmídeos/metabolismo , Transporte Proteico , Retroviridae/metabolismoRESUMO
The Ets2 transcription factor is regulated by mitogen-activated protein (MAP) kinase phosphorylation of a single threonine residue. We generated by gene targeting a single codon mutation in Ets2 substituting Ala for the critical Thr-72 phosphorylation site (Ets2A72), to investigate the importance of MAP kinase activation of Ets2 in embryo and tumor development. Ets2(A72/A72) mice are viable and develop normally. However, combining the Ets2A72 allele with a deletion mutant of Ets2 results in lethality at E11.5 and shows that Ets2A72 is a hypomorphic allele. Mammary tumors caused by transgenic polyomavirus middle T antigen, activated Neu(Erbb2), or the combination of Neu and transgenic VEGF (Neu; VEGF-25) were all restricted in Ets2(A72/A72) females. The Ets2(A72/A72) restriction on Neu; VEGF-25 tumor growth was associated with increased p21Cip1 expression. The size of tumors transplanted into fat pads of mice with Ets2 targeted alleles was correlated directly with Ets2 activity and fewer stromal cells expressing matrix metalloproteinase 9 (MMP-9). Decreased MMP-3 and MMP-9 mRNAs were confirmed in Ets2(A72/A72) macrophages. Activation of Ets2 at Thr-72 acts in the stroma, downstream of vascular endothelial growth factor production, in part through the regulation of macrophage proteases to support the progression of Neu- and polyomavirus middle-T-initiated mammary tumors.
Assuntos
Proteínas de Ligação a DNA , Neoplasias Mamárias Experimentais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Transativadores/metabolismo , Fatores de Transcrição , Alelos , Substituição de Aminoácidos , Animais , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , DNA Complementar/genética , Desenvolvimento Embrionário e Fetal , Feminino , Expressão Gênica , Marcação de Genes , Heterozigoto , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fenótipo , Fosforilação , Gravidez , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.
Assuntos
Carcinoma Intraductal não Infiltrante/prevenção & controle , Proliferação de Células/efeitos dos fármacos , Neoplasias Mamárias Experimentais/prevenção & controle , Lesões Pré-Cancerosas/prevenção & controle , Sirolimo/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/fisiologia , Apoptose/efeitos dos fármacos , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Tomografia por Emissão de Pósitrons , Lesões Pré-Cancerosas/irrigação sanguínea , Lesões Pré-Cancerosas/patologia , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Fatores de TempoRESUMO
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.
Assuntos
Ciclo-Oxigenase 2/deficiência , Neoplasias Mamárias Experimentais/genética , Receptor ErbB-2/genética , Animais , Ciclo-Oxigenase 2/genética , Feminino , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/enzimologia , Neovascularização Patológica/genéticaRESUMO
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.
Assuntos
Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Heterogeneidade Genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Animais , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Linhagem Celular Tumoral , Instabilidade Cromossômica , Aberrações Cromossômicas , Cromossomos de Mamíferos , Feminino , Regulação Neoplásica da Expressão Gênica , Hormônios Gonadais/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Metástase Neoplásica/patologia , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologiaRESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Carcinoma Intraductal não Infiltrante/tratamento farmacológico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Animais , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Quimioprevenção , Progressão da Doença , Feminino , Humanos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Invasividade Neoplásica , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/fisiologiaRESUMO
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.
Assuntos
Carcinoma/patologia , Linhagem Celular Tumoral , Neoplasias Mamárias Experimentais/patologia , Animais , Carcinoma/genética , Carcinoma/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Metástase Neoplásica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores de Estrogênio/análise , Receptores de Estrogênio/metabolismoRESUMO
A transplantable model of human ductal carcinoma in situ that progresses to invasive carcinoma was developed from a genetically engineered mouse (GEM). Additional lines were established using early mammary premalignant lesions from transgenic MMTV-PyV-mT mice. These lines were verified to be premalignant and transplanted repeatedly to establish stable and predictable properties. Here, we report the first in-depth molecular analysis of neoplastic progression occurring in one premalignant transplantable GEM-derived line. Oligonucleotide microarrays showed that many genes are differentially expressed between the quiescent and prelactating mammary gland and the premalignant GEM outgrowth. In contrast, a small but consistent group of genes was associated with the transformation from premalignancy to tumor. This suggests that the majority of gene expression changes occur during the premalignant transition from normal to premalignancy, whereas many fewer changes occur during the malignant transition from premalignancy to invasive carcinoma. The premalignant transition is associated with several cell cycle-related genes and the up-regulation of oncogenes is associated with various cancers (Ccnd11, Cdk4, Myb, and Ect2). The changes identified in the malignant transition included genes previously associated with human breast cancer progression. Misregulation of the insulin-like growth factor and transforming growth factor-beta signaling pathways and the stromal-epithelial interaction were implicated. Our results suggest that this transplantable GEM-based model recapitulates human ductal carcinoma in situ at both histologic and molecular levels. With consistent tumor latency and molecular profiles, this model provides an experimental platform that can be used to assess functional genomics and molecular pharmacology and to test promising chemoprevention strategies.
Assuntos
Carcinoma Ductal/genética , Carcinoma Ductal/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Engenharia Genética , Animais , Carcinoma Ductal/metabolismo , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
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.
Assuntos
Antígenos Transformantes de Poliomavirus/química , Carcinoma in Situ/patologia , Neoplasias Mamárias Animais/patologia , Animais , Carcinoma in Situ/virologia , Diferenciação Celular , Linhagem Celular Tumoral , DNA/metabolismo , Progressão da Doença , Evolução Molecular , Humanos , Imuno-Histoquímica , Pulmão/patologia , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/virologia , Camundongos , Camundongos Transgênicos , Família Multigênica , Metástase Neoplásica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Fenótipo , RNA/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Tempo , TransgenesRESUMO
INTRODUCTION: In order to study metastatic disease, we employed the use of two related polyomavirus middle T transgenic mouse tumor transplant models of mammary carcinoma (termed Met and Db) that display significant differences in metastatic potential. METHODS: Through suppression subtractive hybridization coupled to the microarray, we found osteopontin (OPN) to be a highly expressed gene in the tumors of the metastatic mouse model, and a lowly expressed gene in the tumors of the lowly metastatic mouse model. We further analyzed the role of OPN in this model by examining sense and antisense constructs using in vitro and in vivo methods. RESULTS: With in vivo metastasis assays, the antisense Met cells showed no metastatic tumor formation to the lungs of recipient mice, while wild-type Met cells, with higher levels of OPN, showed significant amounts of metastasis. The Db cells showed a significantly reduced metastasis rate in the in vivo metastasis assay as compared with the Met cells. Db cells with enforced overexpression of OPN showed elevated levels of OPN but did not demonstrate an increase in the rate of metastasis compared with the wild-type Db cells. CONCLUSIONS: We conclude that OPN is an essential regulator of the metastatic phenotype seen in polyomavirus middle T-induced mammary tumors. Yet OPN expression alone is not sufficient to cause metastasis. These data suggest a link between metastasis and phosphatidylinositol-3-kinase-mediated transcriptional upregulation of OPN, but additional phosphatidylinositol-3-kinase-regulated genes may be essential in precipitating the metastasis phenotype in the polyomavirus middle T model.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Sialoglicoproteínas/fisiologia , Animais , Antígenos Transformantes de Poliomavirus/fisiologia , Transformação Celular Neoplásica/genética , Transformação Celular Viral/genética , Feminino , Perfilação da Expressão Gênica , Biblioteca Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Transplante de Neoplasias , Oligodesoxirribonucleotídeos Antissenso/genética , Osteopontina , Fosfatidilinositol 3-Quinases/fisiologia , RNA Complementar/genética , Proteínas Recombinantes de Fusão/fisiologia , Sialoglicoproteínas/genética , Técnica de Subtração , TransfecçãoRESUMO
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.
Assuntos
Neoplasias da Mama/imunologia , Modelos Animais de Doenças , Receptores ErbB/imunologia , Formaldeído/química , Micro-Ondas , Receptores de Estrogênio/imunologia , Receptores de Progesterona/imunologia , Algoritmos , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , CamundongosRESUMO
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.
Assuntos
Neoplasias da Mama/patologia , Adulto , Animais , Medula Óssea/patologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro/farmacologia , Citogenética , Transição Epitelial-Mesenquimal , Éxons , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Cariotipagem , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Fenótipo , PrognósticoRESUMO
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.
Assuntos
Neoplasias Mamárias Experimentais/fisiopatologia , Fatores de Transcrição/fisiologia , Proteína Wnt1/fisiologia , Animais , Sequência de Bases , Primers do DNA , Feminino , Imuno-Histoquímica , Neoplasias Mamárias Experimentais/genética , Vírus do Tumor Mamário do Camundongo , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , Fatores de Transcrição/genética , Transgenes , Proteína Wnt1/genética , beta Catenina/metabolismoRESUMO
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.