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PURPOSE AND METHODS: In human basal-like breast cancer, mutations and deletions in TP53 and BRCA1 are frequent oncogenic events. Thus, we interbred mice expressing the CRE-recombinase with mice harboring loxP sites at TP53 and BRCA1 (K14-Cre; p53f/f Brca1f/f) to test the hypothesis that tissue-specific deletion of TP53 and BRCA1 would give rise to tumors reflective of human basal-like breast cancer. RESULTS: In support of our hypothesis, these transgenic mice developed tumors that express basal-like cytokeratins and demonstrated intrinsic gene expression features similar to human basal-like tumors. Array comparative genomic hybridization revealed a striking conservation of copy number alterations between the K14-Cre; p53f/f Brca1f/f mouse model and human basal-like breast cancer. Conserved events included MYC amplification, KRAS amplification, and RB1 loss. Microarray analysis demonstrated that these DNA copy number events also led to corresponding changes in signatures of pathway activation including high proliferation due to RB1 loss. K14-Cre; p53f/f Brca1f/f also matched human basal-like breast cancer for a propensity to have immune cell infiltrates. Given the long latency of K14-Cre; p53f/f Brca1f/f tumors (~ 250 days), we created tumor syngeneic transplant lines, as well as in vitro cell lines, which were tested for sensitivity to carboplatin and paclitaxel. These therapies invoked acute regression, extended overall survival, and resulted in gene expression signatures of an anti-tumor immune response. CONCLUSION: These findings demonstrate that this model is a valuable preclinical resource for the study of human basal-like breast cancer.
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Modelos Animales de Enfermedad , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Animales , Proteína BRCA1 , Femenino , Humanos , Ratones , Ratones TransgénicosRESUMEN
Exposure to exercise following a breast cancer diagnosis is associated with reductions in the risk of recurrence. However, it is not known whether breast cancers within the same molecular-intrinsic subtype respond differently to exercise. Syngeneic mouse models of claudin-low breast cancer (i.e., EO771, 4TO7, and C3(1)SV40Tag-p16-luc) were allocated to a uniform endurance exercise treatment dose (forced treadmill exercise) or sham-exercise (stationary treadmill). Compared to sham-controls, endurance exercise treatment differentially affected tumor growth rate: 1- slowed (EO771), 2- accelerated (C3(1)SV40Tag-p16-luc), or 3- was not affected (4TO7). Differential sensitivity of the three tumor lines to exercise was paralleled by effects on intratumoral Ki-67, Hif1-α, and metabolic programming. Inhibition of Hif1-α synthesis by the cardiac glycoside, digoxin, completely abrogated exercise-accelerated tumor growth in C3(1)SV40Tag-p16-luc. These results suggest that intratumoral Hif1-α expression is an important determinant of claudin-low breast cancer adaptation to exercise treatment.
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The vast majority of cancer-related deaths are due to metastatic disease, whereby primary tumor cells disseminate and colonize distal sites within the body. Triple negative breast cancer typically displays aberrant Wnt signaling, lacks effective targeted therapies, and compared with other breast cancer subtypes, is more likely to recur and metastasize. We developed a Wnt-driven lung metastasis model of triple negative breast cancer (metM-Wntlung) through serial passaging of our previously described, nonmetastatic, claudin-low M-Wnt cell line. metM-Wntlung cells displayed characteristics of epithelial-to-mesenchymal transition (e.g., increased invasiveness) with some re-epithealization (e.g., increased adhesion, tight colony formation, increased E-cadherin expression, and decreased Vimentin and Fibronectin expression). When orthotopically transplanted into syngeneic mice, metM-Wntlung cells readily formed tumors and metastasized in vivo, and tumor growth and metastasis were enhanced in obese mice compared with non-obese mice. Gene expression analysis revealed several genes and pathways altered in metM-Wntlung cells compared with M-Wnt cells, including multiple genes associated with epithelial-to-mesenchymal transition, energy metabolism and inflammation. Moreover, obesity caused significant transcriptomic changes, especially in metabolic pathways. Metabolic flux analyses showed greater metabolic plasticity, with heightened mitochondrial and glycolytic energetics in metM-Wntlung cells relative to M-Wnt cells. Similar metabolic profiles were found in a second triple negative breast cancer progression series, M6 and M6C cells. These findings suggest that metabolic reprogramming is a feature of metastatic potential in triple negative breast cancer. Thus, targeting metastases-associated metabolic perturbations may represent a novel strategy for reducing the burden of metastatic triple negative breast cancer, particularly in obese women.
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Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Terapia Molecular Dirigida , Proteogenómica , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Femenino , Humanos , Ratones , Fosforilación , Transducción de Señal , Transcriptoma/genéticaRESUMEN
Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges.
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Advances in the screening of new therapeutic options have significantly reduced the breast cancer death rate over the last decade. Despite these advances, breast cancer remains the second leading cause of cancer death among women. This is due in part to the complexity of the disease, which is characterized by multiple subtypes that are driven by different genetic mechanisms and that likely arise from different cell types of origin. Because these differences often drive treatment options and outcomes, it is important to select relevant preclinical model systems to study new therapeutic interventions and tumor biology. Described in this unit are the characteristics and applications of validated genetically engineered mouse models (GEMMs) of basal-like, luminal, and claudin-low human subtypes of breast cancer. These different subtypes have different clinical outcomes and require different treatment strategies. These GEMMs can be considered faithful surrogates of their human disease counterparts. They represent alternative preclinical tumor models to cell line and patient-derived xenografts for preclinical drug discovery and tumor biology studies.
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Animales Modificados Genéticamente , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Descubrimiento de Drogas , Femenino , HumanosRESUMEN
BACKGROUND: Human breast cancer is a heterogeneous disease consisting of multiple molecular subtypes. Genetically engineered mouse models are a useful resource for studying mammary cancers in vivo under genetically controlled and immune competent conditions. Identifying murine models with conserved human tumor features will facilitate etiology determinations, highlight the effects of mutations on pathway activation, and should improve preclinical drug testing. RESULTS: Transcriptomic profiles of 27 murine models of mammary carcinoma and normal mammary tissue were determined using gene expression microarrays. Hierarchical clustering analysis identified 17 distinct murine subtypes. Cross-species analyses using three independent human breast cancer datasets identified eight murine classes that resemble specific human breast cancer subtypes. Multiple models were associated with human basal-like tumors including TgC3(1)-Tag, TgWAP-Myc and Trp53-/-. Interestingly, the TgWAPCre-Etv6 model mimicked the HER2-enriched subtype, a group of human tumors without a murine counterpart in previous comparative studies. Gene signature analysis identified hundreds of commonly expressed pathway signatures between linked mouse and human subtypes, highlighting potentially common genetic drivers of tumorigenesis. CONCLUSIONS: This study of murine models of breast carcinoma encompasses the largest comprehensive genomic dataset to date to identify human-to-mouse disease subtype counterparts. Our approach illustrates the value of comparisons between species to identify murine models that faithfully mimic the human condition and indicates that multiple genetically engineered mouse models are needed to represent the diversity of human breast cancers. The reported trans-species associations should guide model selection during preclinical study design to ensure appropriate representatives of human disease subtypes are used.
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Neoplasias de la Mama/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Transcriptoma/genética , Animales , Neoplasias de la Mama/clasificación , Carcinogénesis/genética , Carcinogénesis/metabolismo , Análisis por Conglomerados , Femenino , Humanos , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia de ADNRESUMEN
PURPOSE: To use genetically engineered mouse models (GEMM) and orthotopic syngeneic murine transplants (OST) to develop gene expression-based predictors of response to anticancer drugs in human tumors. These mouse models offer advantages including precise genetics and an intact microenvironment/immune system. EXPERIMENTAL DESIGN: We examined the efficacy of 4 chemotherapeutic or targeted anticancer drugs, alone and in combination, using mouse models representing 3 distinct breast cancer subtypes: Basal-like (C3(1)-T-antigen GEMM), Luminal B (MMTV-Neu GEMM), and Claudin-low (T11/TP53-/- OST). We expression-profiled tumors to develop signatures that corresponded to treatment and response, and then tested their predictive potential using human patient data. RESULTS: Although a single agent exhibited exceptional efficacy (i.e., lapatinib in the Neu-driven model), generally single-agent activity was modest, whereas some combination therapies were more active and life prolonging. Through analysis of RNA expression in this large set of chemotherapy-treated murine tumors, we identified a pair of gene expression signatures that predicted pathologic complete response to neoadjuvant anthracycline/taxane therapy in human patients with breast cancer. CONCLUSIONS: These results show that murine-derived gene signatures can predict response even after accounting for common clinical variables and other predictive genomic signatures, suggesting that mice can be used to identify new biomarkers for human patients with cancer.
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Animales Modificados Genéticamente , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Biomarcadores Farmacológicos/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/genética , Carboplatino/administración & dosificación , Ciclofosfamida/administración & dosificación , Doxorrubicina/administración & dosificación , Clorhidrato de Erlotinib , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Lapatinib , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Paclitaxel/administración & dosificación , Paclitaxel/farmacocinética , Quinazolinas/administración & dosificación , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genéticaRESUMEN
PURPOSE: Anticancer drug development is inefficient, but genetically engineered murine models (GEMM) and orthotopic, syngeneic transplants (OST) of cancer may offer advantages to in vitro and xenograft systems. EXPERIMENTAL DESIGN: We assessed the activity of 16 treatment regimens in a RAS-driven, Ink4a/Arf-deficient melanoma GEMM. In addition, we tested a subset of treatment regimens in three breast cancer models representing distinct breast cancer subtypes: claudin-low (T11 OST), basal-like (C3-TAg GEMM), and luminal B (MMTV-Neu GEMM). RESULTS: Like human RAS-mutant melanoma, the melanoma GEMM was refractory to chemotherapy and single-agent small molecule therapies. Combined treatment with AZD6244 [mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor] and BEZ235 [dual phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor] was the only treatment regimen to exhibit significant antitumor activity, showed by marked tumor regression and improved survival. Given the surprising activity of the "AZD/BEZ" combination in the melanoma GEMM, we next tested this regimen in the "claudin-low" breast cancer model that shares gene expression features with melanoma. The AZD/BEZ regimen also exhibited significant activity in this model, leading us to testing in even more diverse GEMMs of basal-like and luminal breast cancer. The AZD/BEZ combination was highly active in these distinct breast cancer models, showing equal or greater efficacy compared with any other regimen tested in studies of over 700 tumor-bearing mice. This regimen even exhibited activity in lapatinib-resistant HER2(+) tumors. CONCLUSION: These results show the use of credentialed murine models for large-scale efficacy testing of diverse anticancer regimens and predict that combinations of PI3K/mTOR and MEK inhibitors will show antitumor activity in a wide range of human malignancies.
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Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Neoplasias Mamarias Animales/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Bencimidazoles/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Humanos , Imidazoles/administración & dosificación , Quinasas Quinasa Quinasa PAM/metabolismo , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Melanoma/genética , Melanoma/patología , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Quinolinas/administración & dosificación , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC.
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Neoplasias de la Mama/enzimología , Caveolinas/metabolismo , Transformación Celular Neoplásica/metabolismo , Glicosiltransferasas/metabolismo , Glándulas Mamarias Animales/enzimología , Neoplasias Mamarias Experimentales/enzimología , Células Madre Neoplásicas/enzimología , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio/metabolismo , Caveolinas/genética , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Claudinas/metabolismo , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glicosiltransferasas/deficiencia , Glicosiltransferasas/genética , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/trasplante , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Persona de Mediana Edad , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/trasplante , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-met/genética , Receptor IGF Tipo 1/metabolismo , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transducción de SeñalRESUMEN
Using novel murine models of claudin-low and basal-like breast cancer, we tested the hypothesis that diet-induced obesity (DIO) and calorie restriction (CR) differentially modulate progression of these aggressive breast cancer subtypes. For model development, we characterized two cell lines, "mesenchymal (M)-Wnt" and "epithelial (E)-Wnt," derived from MMTV-Wnt-1 transgenic mouse mammary tumors. M-Wnt, relative to E-Wnt, cells were tumor-initiating cell (TIC)-enriched (62% vs. 2.4% CD44(high)/CD24(low)) and displayed enhanced ALDEFLUOR positivity, epithelial-to-mesenchymal transition (EMT) marker expression, mammosphere-forming ability, migration, invasion, and tumorigenicity (P < 0.001; each parameter). M-Wnt and E-Wnt cells clustered with claudin-low and basal-like breast tumors, respectively, in gene expression profiles and recapitulated these tumors when orthotopically transplanted into ovariectomized C57BL/6 mice. To assess the effects of energy balance interventions on tumor progression and EMT, mice were administered DIO, control, or CR diets for 8 weeks before orthotopic transplantation of M-Wnt or E-Wnt cells (for each cell line, n = 20 mice per diet) and continued on their diets for 6 weeks while tumor growth was monitored. Relative to control, DIO enhanced M-Wnt (P = 0.01), but not E-Wnt, tumor progression; upregulated EMT- and TIC-associated markers including N-cadherin,fibronectin, TGFß, Snail, FOXC2, and Oct4 (P < 0.05, each); and increased intratumoral adipocytes. Conversely, CR suppressed M-Wnt and E-Wnt tumor progression (P < 0.02, each) and inhibited EMT and intratumoral adipocyte accumulation. Thus, dietary energy balance interventions differentially modulate EMT and progression of claudin-low and basal-like tumors. EMT pathway components may represent targets for breaking the obesity-breast cancer link, particularly for preventing and/or controlling TIC-enriched subtypes such as claudin-low breast cancer.
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Carcinoma Basocelular/patología , Claudinas/deficiencia , Dieta , Metabolismo Energético , Transición Epitelial-Mesenquimal , Neoplasias Mamarias Animales/patología , Células Madre Neoplásicas/patología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma Basocelular/etiología , Carcinoma Basocelular/metabolismo , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Neoplasias Mamarias Animales/etiología , Neoplasias Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovariectomía , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Proteínas Quinasas/genética , Proteoma/análisis , Animales , Antineoplásicos/uso terapéutico , Bencenosulfonatos/uso terapéutico , Bencimidazoles/uso terapéutico , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piridinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/genética , SorafenibRESUMEN
BACKGROUND: Cyclin-dependent kinases (CDKs) regulate cell proliferation and coordinate the cell cycle checkpoint response to DNA damage. Although inhibitors with varying selectivity to specific CDK family members have been developed, selective CDK4/6 inhibitors have emerged as the most attractive antineoplastic agents because of the importance of CDK4/6 activity in regulating cell proliferation and the toxic effects associated with inhibition of other CDKs (eg, CDK1 and CDK2). METHODS: FVB/N wild-type mice (n = 13) were used to evaluate carboplatin-induced myelosuppression in bone marrow by complete blood cell counts after treatment with the CDK4/6 inhibitor PD0332991. Genetically engineered murine models of retinoblastoma (Rb)-competent (MMTV-c-neu) and Rb-incompetent (C3-TAg) breast cancer (n = 16 MMTV-c-neu mice in the carboplatin plus vehicle control group, n = 17 MMTV-c-neu mice in the carboplatin plus PD0332991 group, n = 17 C3-TAg mice in the carboplatin plus vehicle control group, and n = 14 C3-TAg mice in the carboplatin plus PD0332991 group) were used to investigate the antitumor activity of PD0332991 alone or in combination with chemotherapy. All statistical tests were two-sided. RESULTS: Coadministration of PD0332991 with carboplatin compared with carboplatin alone in FVB/N wild-type mice increased hematocrit (51.2% vs 33.5%, difference = 17.7%, 95% confidence interval [CI] = -26.7% to -8.6%, P < .001), platelet counts (1321 vs 758.5 thousand cells per µL, difference = 562.5 thousand cells per µL, 95% CI = -902.8 to -222.6, P = .002), myeloid cells (granulocytes and monocytes; 3.1 vs 1.6 thousand cells per µL, difference = 1.5 thousand cells per µL, 95% CI = -2.23 to -0.67, P < .001), and lymphocytes (7.9 vs 5.4 thousand cells per µL, difference = 2.5 thousand cells per µL, 95% CI = -4.75 to -0.18, P = .02). Daily administration of PD0332991 exhibited antitumor activity in MMTV-c-neu mice as a single agent. However, the combination of carboplatin plus PD0332991 decreased antitumor activity compared with carboplatin alone in Rb-competent mice (mean percent change in tumor volume at day 21 = -52.6% vs 3.7% for carboplatin and carboplatin plus PD0332991, respectively, difference = 56.3%, 95% CI = -109.0% to -3.6%, P = .04). In contrast, Rb-deficient tumors in C3-Tag mice were resistant to PD0332991, and coadministration of PD0332991 plus carboplatin had no effect on in vivo tumor growth (mean percent change in tumor volume at day 21 = 118.8% and 109.1% for carboplatin and carboplatin plus PD0332991, respectively, difference = 9.7%, 95% CI = -183.5% to 202.9%, P = .92). Finally, in tumor-bearing mice, coadministration of PD0332991 with carboplatin provided statistically significant protection of platelets (P = .04). CONCLUSION: We believe that the present data support a possible role for CDK4/6 inhibitors in a majority of patients with advanced cancer: to either inhibit tumor growth in CDK4/6-dependent tumors or ameliorate the dose-limiting toxicities of chemotherapy in CDK4/6-indepdendent tumors. Our data also suggest CDK4/6 inhibitors should not be combined with DNA-damaging therapies, such as carboplatin, to treat tumors that require CDK4/6 activity for proliferation.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Apoptosis/efectos de los fármacos , Plaquetas/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carboplatino/farmacología , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Doxorrubicina/farmacología , Activación Enzimática , Femenino , Citometría de Flujo , Ratones , Piperazinas/efectos adversos , Sustancias Protectoras/farmacología , Piridinas/efectos adversos , Receptor ErbB-2/metabolismo , Retinoblastoma/tratamiento farmacológicoRESUMEN
Human breast cancer is a heterogeneous disease composed of different histologies and molecular subtypes, many of which are not replicated in animal models. Here, we report a mouse model of breast cancer that generates unique tumor histologies including tubular, adenosquamous, and lipid-rich carcinomas. Utilizing a nononcogenic variant of polyoma middle T oncogene (PyMT) that requires a spontaneous base-pair deletion to transform cells, in conjunction with lentiviral transduction and orthotopic transplantation of primary mammary epithelial cells, this model sporadically induces oncogene expression in both the luminal and myoepithelial cell lineages of the normal mouse mammary epithelium. Microarray and hierarchical analyses using an intrinsic subtype gene set revealed that lentiviral PyMT generates both luminal and basal-like tumors. Cumulatively, these results show that low-level expression of PyMT in a broad range of cell types significantly increases tumor heterogeneity and establishes a mouse model of several rare human breast cancer subtypes.
RESUMEN
The claudin-low subtype is a recently identified rare molecular subtype of human breast cancer that expresses low levels of tight and adherens junction genes and shows high expression of epithelial-to-mesenchymal transition (EMT) genes. These tumors are enriched in gene expression signatures derived from human tumor-initiating cells (TICs) and human mammary stem cells. Through cross-species analysis, we discovered mouse mammary tumors that have similar gene expression characteristics as human claudin-low tumors and were also enriched for the human TIC signature. Such claudin-low tumors were similarly rare but came from a number of distinct mouse models, including the p53 null transplant model. Here we present a molecular characterization of 50 p53 null mammary tumors compared with other mouse models and human breast tumor subtypes. Similar to human tumors, the murine p53 null tumors fell into multiple molecular subtypes, including two basal-like, a luminal, a claudin-low, and a subtype unique to this model. The claudin-low tumors also showed high gene expression of EMT inducers, low expression of the miR-200 family, and low to absent expression of both claudin 3 and E-cadherin. These murine subtypes also contained distinct genomic DNA copy number changes, some of which are similarly altered in their cognate human subtype counterpart. Finally, limiting dilution transplantation revealed that p53 null claudin-low tumors are highly enriched for TICs compared with the more common adenocarcinomas arising in the same model, thus providing a unique preclinical mouse model to investigate the therapeutic response of TICs.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Genómica/métodos , Neoplasias Mamarias Animales/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Adenocarcinoma/clasificación , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Neoplasias de la Mama/clasificación , Claudinas/metabolismo , Análisis por Conglomerados , Variaciones en el Número de Copia de ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias/genética , Genoma Humano/genética , Humanos , Neoplasias Mamarias Animales/clasificación , Neoplasias Mamarias Animales/patología , Ratones , MicroARNs/metabolismo , Trasplante de Neoplasias , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Tacrolimus (FK506) is an immunosuppressive drug that binds to the immunophilin FKBPB12. The FK506-FKBP12 complex associates with calcineurin and inhibits its phosphatase activity, resulting in inhibition of nuclear translocation of nuclear factor of activated T-cells (NFAT). There is increasing data supporting a critical role of NFAT in mediating angiogenic responses stimulated by both vascular endothelial growth factor (VEGF) and a novel angiogenesis factor, secreted frizzled-related protein 2 (SFRP2). Since both VEGF and SFRP2 are expressed in breast carcinomas, we hypothesized that tacrolimus would inhibit breast carcinoma growth. Using IHC (IHC) with antibodies to FKBP12 on breast carcinomas we found that FKBP12 localizes to breast tumor vasculature. Treatment of MMTV-neu transgenic mice with tacrolimus (3 mg/kg i.p. daily) (nâ=â19) resulted in a 73% reduction in the growth rate for tacrolimus treated mice compared to control (nâ=â15), pâ=â0.003; which was associated with an 82% reduction in tumor microvascular density (p<0.001) by IHC. Tacrolimus (1 µM) inhibited SFRP2 induced endothelial tube formation by 71% (pâ=â0.005) and inhibited VEGF induced endothelial tube formation by 67% (pâ=â0.004). To show that NFATc3 is required for SFRP2 stimulated angiogenesis, NFATc3 was silenced with shRNA in endothelial cells. Sham transfected cells responded to SFRP2 stimulation in a tube formation assay with an increase in the number of branch points (p<0.003), however, cells transfected with shRNA to NFATc3 showed no increase in tube formation in response to SFRP2. This demonstrates that NFATc3 is required for SFRP2 induced tube formation, and tacrolimus inhibits angiogenesis in vitro and breast carcinoma growth in vivo. This provides a rationale for examining the therapeutic potential of tacrolimus at inhibiting breast carcinoma growth in humans.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Inhibidores de la Calcineurina , Calcineurina/metabolismo , Proteínas de la Membrana/farmacología , Factores de Transcripción NFATC/metabolismo , Neovascularización Patológica/inducido químicamente , Tacrolimus/farmacología , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Transgénicos , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Factores de Transcripción NFATC/deficiencia , Factores de Transcripción NFATC/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Interferencia de ARN , Tacrolimus/uso terapéutico , Proteína 1A de Unión a Tacrolimus/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , beta Catenina/deficiencia , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Mammary epithelia are composed of luminal and myoepithelial/basal cells whose neoplastic transformations lead to distinct types of breast cancers with diverse clinical features. We report that mice deficient for the CDK4/6 inhibitor p18(Ink4c) spontaneously develop ER-positive luminal tumors at a high penetrance. Ink4c deletion stimulates luminal progenitor cell proliferation at pubertal age and maintains an expanded luminal progenitor cell population throughout life. We demonstrate that GATA3 binds to and represses INK4C transcription. In human breast cancers, low INK4C and high GATA3 expressions are simultaneously observed in luminal A type tumors and predict a favorable patient outcome. Hence, p18(INK4C) is a downstream target of GATA3, constrains luminal progenitor cell expansion, and suppresses luminal tumorigenesis in the mammary gland.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Transformación Celular Neoplásica , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Animales , Neoplasias de la Mama/genética , Diferenciación Celular , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Ratones , Ratones Endogámicos BALB C , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
BACKGROUND: Although numerous mouse models of breast carcinomas have been developed, we do not know the extent to which any faithfully represent clinically significant human phenotypes. To address this need, we characterized mammary tumor gene expression profiles from 13 different murine models using DNA microarrays and compared the resulting data to those from human breast tumors. RESULTS: Unsupervised hierarchical clustering analysis showed that six models (TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3, TgWAP-Tag, and TgC3(1)-Tag) yielded tumors with distinctive and homogeneous expression patterns within each strain. However, in each of four other models (TgWAP-T121, TgMMTV-Wnt1, Brca1Co/Co;TgMMTV-Cre;p53+/- and DMBA-induced), tumors with a variety of histologies and expression profiles developed. In many models, similarities to human breast tumors were recognized, including proliferation and human breast tumor subtype signatures. Significantly, tumors of several models displayed characteristics of human basal-like breast tumors, including two models with induced Brca1 deficiencies. Tumors of other murine models shared features and trended towards significance of gene enrichment with human luminal tumors; however, these murine tumors lacked expression of estrogen receptor (ER) and ER-regulated genes. TgMMTV-Neu tumors did not have a significant gene overlap with the human HER2+/ER- subtype and were more similar to human luminal tumors. CONCLUSION: Many of the defining characteristics of human subtypes were conserved among the mouse models. Although no single mouse model recapitulated all the expression features of a given human subtype, these shared expression features provide a common framework for an improved integration of murine mammary tumor models with human breast tumors.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/genética , Animales , Neoplasias de la Mama/patología , Análisis por Conglomerados , Perfilación de la Expresión Génica , Humanos , Neoplasias Mamarias Experimentales/patología , Ratones , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de la EspecieRESUMEN
PURPOSE: The prognosis of a patient with estrogen receptor (ER) and/or progesterone receptor (PR) -positive breast cancer can be highly variable. Therefore, we developed a gene expression-based outcome predictor for ER+ and/or PR+ (ie, luminal) breast cancer patients using biologic differences among these tumors. MATERIALS AND METHODS: The ER+ MCF-7 breast cancer cell line was treated with 17beta-estradiol to identify estrogen-regulated genes. These genes were used to develop an outcome predictor on a training set of 65 luminal epithelial primary breast carcinomas. The outcome predictor was then validated on three independent published data sets. Results The estrogen-induced gene set identified in MCF-7 cells was used to hierarchically cluster a 65 tumor training set into two groups, which showed significant differences in survival (P = .0004). Supervised analyses identified 822 genes that optimally defined these two groups, with the poor-prognosis group IIE showing high expression of cell proliferation and antiapoptosis genes. The good prognosis group IE showed high expression of estrogen- and GATA3-regulated genes. Mean expression profiles (ie, centroids) created for each group were applied to ER+ and/or PR+ tumors from three published data sets. For all data sets, Kaplan-Meier survival analyses showed significant differences in relapse-free and overall survival between group IE and IIE tumors. Multivariate Cox analysis of the largest test data set showed that this predictor added significant prognostic information independent of standard clinical predictors and other gene expression-based predictors. CONCLUSION: This study provides new biologic information concerning differences within hormone receptor-positive breast cancers and a means of predicting long-term outcomes in tamoxifen-treated patients.