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1.
Nature ; 508(7494): 103-107, 2014 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-24670641

RESUMEN

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)--a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)--is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44(high)CD24(low) population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Antígeno CD24/metabolismo , Hipoxia de la Célula/genética , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Silenciador del Gen , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Pronóstico , ARN Polimerasa II/metabolismo , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genética , Respuesta de Proteína Desplegada , Proteína 1 de Unión a la X-Box
2.
J Biol Chem ; 291(45): 23756-23768, 2016 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-27650498

RESUMEN

Bromodomain and extraterminal (BET) proteins are epigenetic "readers" that recognize acetylated histones and mark areas of the genome for transcription. BRD4, a BET family member protein, has been implicated in a number of types of cancer, and BET protein inhibitors (BETi) are efficacious in many preclinical cancer models. However, the drivers of response to BETi vary depending on tumor type, and little is known regarding the target genes conveying BETi activity in triple-negative breast cancer (TNBC). Here, we show that BETi repress growth of multiple in vitro and in vivo models of TNBC by inducing two terminal responses: apoptosis and senescence. Unlike in other cancers, response to BETi in TNBC is not dependent upon suppression of MYC Instead, both end points are preceded by the appearance of polyploid cells caused by the suppression of Aurora kinases A and B (AURKA/B), which are critical mediators of mitosis. In addition, AURKA/B inhibitors phenocopy the effects of BETi. These results indicate that Aurora kinases play an important role in the growth suppressive activity of BETi in TNBC. Elucidating the mechanism of response to BETi in TNBC should 1) facilitate the prediction of how distinct TNBC tumors will respond to BETi and 2) inform the rational design of drug combination therapies.


Asunto(s)
Antineoplásicos/uso terapéutico , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa B/antagonistas & inhibidores , Mama/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Factores de Transcripción/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Aurora Quinasa A/metabolismo , Aurora Quinasa B/metabolismo , Mama/metabolismo , Mama/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología
4.
Breast Cancer Res ; 18(1): 6, 2016 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-26757880

RESUMEN

BACKGROUND: Developing novel strategies against treatment-resistant triple negative breast cancer (TNBC) cells remains a significant challenge. The ErbB family, including epidermal growth factor receptor (EGFR), plays key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSC) which are believed to be responsible for tumor initiation and maintenance. Ixabepilone is a new generation microtubule-stabilizing agent, which has been expected to be more efficacious than conventional taxanes. Here we aim to investigate whether the EGFR monoclonal antibody Cetuximab, in combination with Ixabepilone, is more effective in eliminating CSC populations compared to chemotherapy alone in TNBC. METHODS: Representative TNBC cell lines (MDA-MB-231 and SUM159) were used to evaluate breast CSC populations. We used fluorescence-activated cell sorter analysis (CD44(+) and CD24(-/low), or Aldefluor(+)) and a self-renewal assay called mammosphere formation efficiency (MSFE) to measure CSC population size after treatment with Cetuximab, or Cetuximab plus Ixabepilone in vitro. RESULTS: Although there was no significant decrease in cell viability, Cetuximab reduced MSFE and the CSC population in breast cancer cells in vitro and in vivo through inhibition of autophagy. Also, SUM159 and MDA-MB-231 orthotopic tumors demonstrated partial response to Centuximab or Ixabepilone monotherapy; however, the effect of the combination treatment was significant only in SUM159 tumors (p <0.0001), when compared to Ixabepilone alone. CONCLUSIONS: Overall, our findings demonstrate that EGFR-targeted therapy by Cetuximab effectively reduces the CSC population in TNBC tumors. However, combination therapy with Ixabepilone may be effective only in a small subset of TNBCs, warranting further investigation of alternative approaches to target multiple pathways for TNBC treatment.


Asunto(s)
Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Cetuximab/administración & dosificación , Epotilonas/administración & dosificación , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología
5.
Breast Cancer Res ; 17: 25, 2015 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-25849745

RESUMEN

INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. METHODS: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models. RESULTS: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor ß was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal. CONCLUSIONS: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.


Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/metabolismo , Factor de Transcripción Activador 3/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico , Transición Epitelial-Mesenquimal/genética , Femenino , Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Terapia Molecular Dirigida , Invasividad Neoplásica , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Pronóstico , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Stem Cells ; 32(9): 2309-23, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24809620

RESUMEN

Triple negative breast cancer (TNBC) is known to contain a high percentage of CD44(+) /CD24(-/low) cancer stem cells (CSCs), corresponding with a poor prognosis despite systemic chemotherapy. Chloroquine (CQ), an antimalarial drug, is a lysotropic reagent which inhibits autophagy. CQ was identified as a potential CSC inhibitor through in silico gene expression signature analysis of the CD44(+) /CD24(-/low) CSC population. Autophagy plays a critical role in adaptation to stress conditions in cancer cells, and is related with drug resistance and CSC maintenance. Thus, the objectives of this study were to examine the potential enhanced efficacy arising from addition of CQ to standard chemotherapy (paclitaxel) in TNBC and to identify the mechanism by which CQ eliminates CSCs in TNBCs. Herein, we report that CQ sensitizes TNBC cells to paclitaxel through inhibition of autophagy and reduces the CD44(+) /CD24(-/low) CSC population in both preclinical and clinical settings. Also, we are the first to report a mechanism by which CQ regulates the CSCs in TNBC through inhibition of the Janus-activated kinase 2 (Jak2)-signal transducer and activator of transcription 3 signaling pathway by reducing the expression of Jak2 and DNA methyltransferase 1.


Asunto(s)
Cloroquina/farmacología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Janus Quinasa 2/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1 , Femenino , Humanos , Ratones , Ratones Desnudos , Células Madre Neoplásicas/patología , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/metabolismo
7.
Proc Natl Acad Sci U S A ; 109(46): 18707-12, 2012 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-23112172

RESUMEN

Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.


Asunto(s)
Separación Celular/instrumentación , Separación Celular/métodos , Técnicas Analíticas Microfluídicas , Células Madre Neoplásicas/citología , Esferoides Celulares/citología , Línea Celular Tumoral , Humanos
8.
Small ; 10(13): 2688-96, 2014 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-24639392

RESUMEN

Hybrid PET/MRI scanners have the potential to provide fundamental molecular, cellular, and anatomic information essential for optimizing therapeutic and surgical interventions. However, their full utilization is currently limited by the lack of truly multi-modal contrast agents capable of exploiting the strengths of each modality. Here, we report on the development of long-circulating positron-emitting magnetic nanoconstructs (PEM) designed to image solid tumors for combined PET/MRI. PEMs are synthesized by a modified nano-precipitation method mixing poly(lactic-co-glycolic acid) (PLGA), lipids, and polyethylene glycol (PEG) chains with 5 nm iron oxide nanoparticles (USPIOs). PEM lipids are coupled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and subsequently chelated to (64)Cu. PEMs show a diameter of 140 ± 7 nm and a transversal relaxivity r2 of 265.0 ± 10.0 (mM × s)(-1), with a r2/r1 ratio of 123. Using a murine xenograft model bearing human breast cancer cell line (MDA-MB-231), intravenously administered PEMs progressively accumulate in tumors reaching a maximum of 3.5 ± 0.25% ID/g tumor at 20 h post-injection. Correlation of PET and MRI signals revealed non-uniform intratumoral distribution of PEMs with focal areas of accumulation at the tumor periphery. These long-circulating PEMs with high transversal relaxivity and tumor accumulation may allow for detailed interrogation over multiple scales in a clinically relevant setting.


Asunto(s)
Electrones , Imagen por Resonancia Magnética , Magnetismo , Tomografía de Emisión de Positrones , Animales , Ratones , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/patología
9.
Breast Cancer Res ; 15(1): 201, 2013 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-23339383

RESUMEN

Despite improved detection and reduction of breast cancer-related deaths over the recent decade, breast cancer remains the second leading cause of cancer death for women in the US, with 39,510 women expected to succumb to metastatic disease in 2012 alone (American Cancer Society, Cancer Facts &Figures 2012. Atlanta: American Cancer Society; 2012). Continued efforts in classification of breast cancers based on gene expression profiling and genomic sequencing have revealed an underlying complexity and molecular heterogeneity within the disease that continues to challenge therapeutic interventions. To successfully identify and translate new treatment regimens to the clinic, it is imperative that our preclinical models recapitulate this complexity and heterogeneity. In this review article, we discuss the recent advances in development and classification of patient-derived human breast tumor xenograft models that have the potential to facilitate the next phase of drug discovery for personalized cancer therapy based on the unique driver signaling pathways in breast tumor subtypes.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Medicina de Precisión , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias de la Mama/patología , Femenino , Humanos , Patología Molecular , Pacientes , Estados Unidos
10.
Breast Cancer Res ; 15(5): R77, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24008095

RESUMEN

INTRODUCTION: We hypothesized that cells present in normal tissue that bear cancer stem cell markers may represent a cancer cell of origin or a microenvironment primed for tumor development, and that their presence may correlate with the clinically defined subtypes of breast cancer that show increased tumorigenicity and stem cell features. methods: Normal tissues sampled at least 5 cm from primary tumors (normal adjacent tissue) were obtained from 61 chemotherapy-naive patients with breast cancer treated with mastectomy. Samples were stained simultaneously with immunofluorescence for CD44/CD49f/CD133/2 stem cell markers. We assessed the association between CD44+CD49f+CD133/2+ staining in normal adjacent tissue and breast cancer receptor subtype (defined by the expression of the estrogen (ER), progesterone (PR), or human epidermal growth factor-2 (Her2) receptors). We also examined the correlation between CD44+CD49f+CD133/2+ immunofluorescence and each of two previously published gene signatures, one derived from stem-cell enriched tissue and one from BRCA mutated tissue expected to have defective DNA repair. RESULTS: Patients with triple negative breast cancer (ER­/PR­/HER2­) expressed CD44+CD49f+CD133/2+ in 9 of 9 normal adjacent tissue samples compared with 7 of 52 ER+ and/or Her2+ tumors (P < 0.001). Further, expression of CD44+CD49f+CD133/2+ by normal adjacent tissue correlated positively with a stem cell-derived tumorigenic signature (P <0.001) and inversely with a defective DNA-repair signature (P <0.001). CONCLUSION: Normal cells bearing cancer stem cell markers are associated with the triple negative receptor subtype of breast cancer. This study suggests stem cell staining and gene expression signatures from normal breast tissues represent novel tissue-based risk biomarkers for triple negative breast cancer. Validation of these results in additional studies of normal tissue from cancer-free women could lay the foundation for future targeted triple negative breast cancer prevention strategies.


Asunto(s)
Reparación del ADN , Células Madre Neoplásicas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Antígeno AC133 , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Superficie , Biomarcadores/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Factores Epidemiológicos , Femenino , Perfilación de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Glándulas Mamarias Humanas/metabolismo , Péptidos/genética , Péptidos/metabolismo , Neoplasias de la Mama Triple Negativas/mortalidad
11.
New J Phys ; 15: 55004, 2013 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-24039540

RESUMEN

Heterogeneities in the perfusion of solid tumors prevent optimal delivery of nanotherapeutics. Clinical imaging protocols to obtain patient-specific data have proven difficult to implement. It is challenging to determine which perfusion features hold greater prognostic value and to relate measurements to vessel structure and function. With the advent of systemically administered nanotherapeutics, whose delivery is dependent on overcoming diffusive and convective barriers to transport, such knowledge is increasingly important. We describe a framework for the automated evaluation of vascular perfusion curves measured at the single vessel level. Primary tumor fragments, collected from triple-negative breast cancer patients and grown as xenografts in mice, were injected with fluorescence contrast and monitored using intravital microscopy. The time to arterial peak and venous delay, two features whose probability distributions were measured directly from time-series curves, were analyzed using a Fuzzy C-mean (FCM) supervised classifier in order to rank individual tumors according to their perfusion characteristics. The resulting rankings correlated inversely with experimental nanoparticle accumulation measurements, enabling modeling of nanotherapeutics delivery without requiring any underlying assumptions about tissue structure or function, or heterogeneities contained within. With additional calibration, these methodologies may enable the study of nanotherapeutics delivery strategies in a variety of tumor models.

12.
Breast Cancer Res Treat ; 132(3): 993-1000, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21750966

RESUMEN

Obesity is thought to contribute to worse disease outcome in breast cancer as a result of increased levels of adipocyte-secreted endocrine factors, insulin, and insulin-like growth factors (IGFs) that accelerate tumor cell proliferation and impair treatment response. We examined the effects of patient obesity on primary breast tumor gene expression, by profiling transcription of a set of 103 tumors for which the patients' body mass index (BMI) was ascertained. Sample profiles were stratified according to patients' obesity phenotype defined as normal (BMI < 25), overweight (BMI 25-29.9), or obese (BMI ≥ 30). Widespread gene expression alterations were evident in breast tumors from obese patients as compared to other tumors, allowing us to define an obesity-associated cancer transcriptional signature of 662 genes. In multiple public expression data sets of breast cancers (representing > 1,500 patients), manifestation of the obesity signature patterns correlated with manifestation of a gene signature for IGF signaling and (to a lesser extent) with lower levels of estrogen receptor. In one patient cohort, manifestation of the obesity signature correlated with shorter time to metastases. A number of small molecules either induced or suppressed the obesity-associated transcriptional program in vitro; estrogens alpha-estradiol, levonorgestrel, and hexestrol induced the program, while several anti-parkinsonian agents targeting neurotransmitter receptor pathways repressed the program. Obesity in breast cancer patients appears to impact the gene expression patterns of the tumor (perhaps as a result of altered body chemistry). These results warrant further investigation of obesity-associated modifiers of breast cancer risk and disease outcome.


Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/metabolismo , Perfilación de la Expresión Génica , Obesidad/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , Estimación de Kaplan-Meier , Obesidad/complicaciones , Obesidad/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Receptores de Estrógenos/metabolismo , Valores de Referencia , Transducción de Señal , Estadísticas no Paramétricas , Transcripción Genética
13.
Mol Cancer ; 9: 180, 2010 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-20615238

RESUMEN

BACKGROUND: Since the first suggestion of prospectively identifiable cancer stem cells in solid tumors, efforts have been made to characterize reported cancer stem cell surrogates in existing cancer cell lines, and cell lines rich with these surrogates have been used to screen for cancer stem cell targeted agents. Although 293T cells were derived from human embryonic kidney, transplantation of these cells into the mammary fat pad yields aggressive tumors that self-renew as evidenced by serial xenograft passages through transplantation. Herein we fully characterize cancer stem cell-like features in 293T human embryonic kidney cells. RESULTS: 293T cells can be readily cultured and passaged as spheres in serum-free stem cell promoting culture conditions. Cells cultured in vitro as three-dimensional spheres (3D) were shown to contain higher ALDH1 and CD44+/CD24- population compared to monolayer cells. These cells were also resistant to radiation and upregulate stem cell survival signaling including beta-catenin, Notch1 and Survivin in response to radiation. Moreover, 3D spheres generated from the 293T cells have increased expression of mesenchymal genes including vimentin, n-cadherin, zeb1, snail and slug as well as pro-metastatic genes RhoC, Tenascin C and MTA1. In addition, microRNAs implicated in self-renewal and metastases were markedly reduced in 3D spheres. CONCLUSIONS: 293T cells exhibit a cancer stem cell-like phenotype when cultured as 3D spheres and represent an important research tool for studying the molecular and biological mechanisms of cancer stem cells and for testing and developing novel targets for cancer therapy.


Asunto(s)
Riñón/embriología , Neoplasias/patología , Células Madre Neoplásicas/patología , Línea Celular , Medio de Cultivo Libre de Suero , Humanos , Riñón/citología , MicroARNs/genética , Metástasis de la Neoplasia/patología , Estudios Prospectivos , Regulación hacia Arriba
14.
Mol Cancer Ther ; 6(8): 2188-97, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17699716

RESUMEN

Amplification of the HER2 (ErbB2, c-Neu) proto-oncogene in breast cancer is associated with poor prognosis and high relapse rates. HER2/ErbB2, in conjunction with ErbB3, signals through the Akt/phosphatidylinositol 3-kinase pathway and leads to the activation of mammalian target of rapamycin (mTOR), a critical mRNA translation regulator that controls cell growth. Gene expression analysis of mammary tumors collected from mouse mammary tumor virus-c-Neu transgenic mice revealed that mRNA levels of several mTOR pathway members were either up-regulated (p85/phosphatidylinositol 3-kinase and p70S6 kinase) or down-regulated (eIF-4E-BP1) in a manner expected to enhance signaling through this pathway. Treatment of these mice with the mTOR inhibitor rapamycin caused growth arrest and regression of primary tumors with no evidence of weight loss or generalized toxicity. The treatment effects were due to decreased proliferation, associated with reduced cyclin D1 expression, and increased cell death in primary tumors. Whereas many of the dead epithelial cells had the histopathologic characteristics of ischemic necrosis, rapamycin treatment was not associated with changes in microvascular density or apoptosis. Rapamycin also inhibited cellular proliferation in lung metastases. In summary, data from this preclinical model of ErbB2/Neu-induced breast cancer show that inhibition of the mTOR pathway with rapamycin blocks multiple stages of ErbB2/Neu-induced tumorigenic progression.


Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neoplasias Mamarias Experimentales/patología , Receptor ErbB-2/metabolismo , Sirolimus/farmacología , Animales , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Activación Enzimática/efectos de los fármacos , Epitelio/patología , Hiperplasia , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/enzimología , Virus del Tumor Mamario del Ratón , Ratones , Ratones Transgénicos , Proteínas Quinasas/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR
15.
Stem Cell Reports ; 10(1): 212-227, 2018 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-29249663

RESUMEN

Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy.


Asunto(s)
Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Receptores de Leptina/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones SCID , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/patología , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores de Leptina/genética , Elementos de Respuesta , Factor de Transcripción STAT3/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología
16.
Oncogene ; 24(33): 5173-90, 2005 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-15897883

RESUMEN

Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.


Asunto(s)
Perfilación de la Expresión Génica , Neoplasias Mamarias Animales/genética , Lesiones Precancerosas/genética , Receptor ErbB-2/fisiología , Factor de Crecimiento Transformador beta/fisiología , Receptores de Activinas/metabolismo , Animales , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/metabolismo , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Lesiones Precancerosas/metabolismo , Transducción de Señal , Proteína Smad2 , Transactivadores/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador beta/biosíntesis , Regulación hacia Arriba
17.
Sci Transl Med ; 8(334): 334ra53, 2016 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-27075627

RESUMEN

Amplifications at 9p24 have been identified in breast cancer and other malignancies, but the genes within this locus causally associated with oncogenicity or tumor progression remain unclear. Targeted next-generation sequencing of postchemotherapy triple-negative breast cancers (TNBCs) identified a group of 9p24-amplified tumors, which contained focal amplification of the Janus kinase 2 (JAK2) gene. These patients had markedly inferior recurrence-free and overall survival compared to patients with TNBC without JAK2 amplification. Detection of JAK2/9p24 amplifications was more common in chemotherapy-treated TNBCs than in untreated TNBCs or basal-like cancers, or in other breast cancer subtypes. Similar rates of JAK2 amplification were confirmed in patient-derived TNBC xenografts. In patients for whom longitudinal specimens were available, JAK2 amplification was selected for during neoadjuvant chemotherapy and eventual metastatic spread, suggesting a role in tumorigenicity and chemoresistance, phenotypes often attributed to a cancer stem cell-like cell population. In TNBC cell lines with JAK2 copy gains or amplification, specific inhibition of JAK2 signaling reduced mammosphere formation and cooperated with chemotherapy in reducing tumor growth in vivo. In these cells, inhibition of JAK1-signal transducer and activator of transcription 3 (STAT3) signaling had little effect or, in some cases, counteracted JAK2-specific inhibition. Collectively, these results suggest that JAK2-specific inhibitors are more efficacious than dual JAK1/2 inhibitors against JAK2-amplified TNBCs. Furthermore, JAK2 amplification is a potential biomarker for JAK2 dependence, which, in turn, can be used to select patients for clinical trials with JAK2 inhibitors.


Asunto(s)
Cromosomas Humanos Par 9/genética , Amplificación de Genes , Sitios Genéticos , Janus Quinasa 2/genética , Neoplasias de la Mama Triple Negativas/enzimología , Neoplasias de la Mama Triple Negativas/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estudios de Cohortes , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Persona de Mediana Edad , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología
18.
Endocrinology ; 143(9): 3671-80, 2002 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12193583

RESUMEN

Many risk factors for breast cancer are associated with hormonally regulated events. Although numerous mouse models of mammary cancer exist, few address the roles of hormones in spontaneous tumor formation. Here we report that transgenic mice that overexpress LH, resulting in ovarian hyperstimulation, undergo precocious mammary gland development. A significant increase in proliferation leads to ovary-dependent mammary gland hyperplasia. Transgenic glands morphologically mimic those of wild-type pregnant mice and expression levels of multiple milk protein genes are comparable with what is observed at d 14 of pregnancy. In addition to sustained hyperplasia, spontaneous mammary tumors were observed with a mean latency of 41 wk, indicating that chronic hormonal stimulation causes mammary cancer. Although hormonally induced, these tumors lack expression of progesterone receptor, suggesting that following initiating events, the tumors may become hormone independent. This mouse model likely holds great potential as a tool for discovery of hormone-mediated mechanisms of breast cancer and identification of future targets for breast cancer prevention and treatment.


Asunto(s)
Expresión Génica , Hormona Luteinizante/genética , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/etiología , Ovario/efectos de los fármacos , Animales , Femenino , Hiperplasia , Hormona Luteinizante/farmacología , Neoplasias Mamarias Animales/química , Ratones , Ratones Transgénicos , Proteínas de la Leche/genética , Ovario/fisiopatología , Fenotipo , Embarazo , Receptores de Progesterona/análisis , Factores de Riesgo
19.
Cell Res ; 24(5): 542-59, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24675532

RESUMEN

Combinatorial targeted therapies are more effective in treating cancer by blocking by-pass mechanisms or inducing synthetic lethality. However, their clinical application is hampered by resistance and toxicity. To meet this important challenge, we developed and tested a novel concept of biomarker-guided sequential applications of various targeted therapies using ErbB2-overexpressing/PTEN-low, highly aggressive breast cancer as our model. Strikingly, sustained activation of ErbB2 and downstream pathways drives trastuzumab resistance in both PTEN-low/trastuzumab-resistant breast cancers from patients and mammary tumors with intratumoral heterogeneity from genetically-engineered mice. Although lapatinib initially inhibited trastuzumab-resistant mouse tumors, tumors by-passed the inhibition by activating the PI3K/mTOR signaling network as shown by the quantitative protein arrays. Interestingly, activation of the mTOR pathway was also observed in neoadjuvant lapatinib-treated patients manifesting lapatinib resistance. Trastuzumab + lapatinib resistance was effectively overcome by sequential application of a PI3K/mTOR dual kinase inhibitor (BEZ235) with no significant toxicity. However, our p-RTK array analysis demonstrated that BEZ235 treatment led to increased ErbB2 expression and phosphorylation in genetically-engineered mouse tumors and in 3-D, but not 2-D, culture, leading to BEZ235 resistance. Mechanistically, we identified ErbB2 protein stabilization and activation as a novel mechanism of BEZ235 resistance, which was reversed by subsequent treatment with lapatinib + BEZ235 combination. Remarkably, this sequential application of targeted therapies guided by biomarker changes in the tumors rapidly evolving resistance doubled the life-span of mice bearing exceedingly aggressive tumors. This fundamentally novel approach of using targeted therapies in a sequential order can effectively target and reprogram the signaling networks in cancers evolving resistance during treatment.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Mama/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Imidazoles/farmacología , Lapatinib , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Quinolinas/farmacología , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Trastuzumab , Células Tumorales Cultivadas
20.
Stem Cell Reports ; 2(1): 78-91, 2014 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-24511467

RESUMEN

Previous studies have suggested that breast cancer stem cells (BCSCs) mediate metastasis, are resistant to radiation and chemotherapy, and contribute to relapse. Although several BCSC markers have been described, it is unclear whether these markers identify the same or independent BCSCs. Here, we show that BCSCs exist in distinct mesenchymal-like (epithelial-mesenchymal transition [EMT]) and epithelial-like (mesenchymal-epithelial transition [MET]) states. Mesenchymal-like BCSCs characterized as CD24(-)CD44(+) are primarily quiescent and localized at the tumor invasive front, whereas epithelial-like BCSCs express aldehyde dehydrogenase (ALDH), are proliferative, and are located more centrally. The gene-expression profiles of mesenchymal-like and epithelial-like BCSCs are remarkably similar across different molecular subtypes of breast cancer, and resemble those of distinct basal and luminal stem cells found in the normal breast. We propose that the plasticity of BCSCs that allows them to transition between EMT- and MET-like states endows these cells with the capacity for tissue invasion, dissemination, and growth at metastatic sites.


Asunto(s)
Transición Epitelial-Mesenquimal , Células Madre Neoplásicas/citología , Aldehído Deshidrogenasa/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Antígeno CD24/metabolismo , Células Epiteliales/citología , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Células MCF-7 , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Neoplásicas/metabolismo , Transcriptoma
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