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Background Analysis of how digital breast tomosynthesis (DBT) screening affects consecutive screening performance is important to estimate its future value in screening. Purpose To evaluate whether DBT contributes to early detection of breast cancer by assessing cancer detection rates (CDRs), including the fraction of invasive cancers and cancer subtypes in consecutive routine digital mammography (DM). Materials and Methods The paired prospective Malmö Breast Tomosynthesis Screening Trial (MBTST) was performed between January 2010 and February 2015. Participating women underwent one-view DBT and two-view DM at one screening occasion. In this secondary analysis, women were followed up through their first (DM1) and second (DM2) consecutive two-view DM screening rounds after MBTST participation. Cancer diagnoses were identified by referencing records. A logistic regression model, adjusted for age, was used to calculate the odds of luminal A-like cancers with use of the MBTST as reference. Results There were 14 848 final participants in the MBTST (median age, 57 years [IQR, 49-65 years]). Of those, 12 876 women were screened in DM1 (median age, 58 years [IQR, 50-66 years]) and 10 883 were screened in DM2 (median age, 59 years [IQR, 51-67 years]). Compared with CDRs in the trial of 6.5 of 1000 women (95% CI: 5.2, 7.9) for DM and 8.7 of 1000 women (95% CI: 7.3, 10.3) for DBT, the CDR was lower in DM1 (4.6 of 1000 women [95% CI: 3.6, 5.9]) and DM2 (5.3 of 1000 women [95% CI: 4.1, 6.9]). The proportion of invasive cancers was 84.9% (118 of 139 cancers) in the MBTST; the corresponding numbers were 66% (39 of 59 cancers) for DM1 and 83% (50 of 60 cancers) for DM2. The odds of luminal A-like cancers were lower in DM1 at 0.28 (95% CI: 0.12, 0.66 [P = .004]) but not in DM2 at 0.80 (95% CI: 0.40, 1.58 [P = .52]) versus screening in the MBTST. Conclusion CDR and the fraction of invasive cancers were lower in DM1 and then increased in DM2 following the MBTST, indicating earlier cancer detection mainly due to increased detection of luminal A-like cancers in DBT screening. Clinical trials registration no. NCT01091545 © RSNA, 2024 See also the editorial by Hooley and Philpotts in this issue.
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Neoplasias de la Mama , Detección Precoz del Cáncer , Mamografía , Anciano , Femenino , Humanos , Persona de Mediana Edad , Mama/diagnóstico por imagen , Neoplasias de la Mama/diagnóstico por imagen , Detección Precoz del Cáncer/métodos , Mamografía/métodos , Tamizaje Masivo/métodos , Estudios Prospectivos , SueciaRESUMEN
BACKGROUND: Breast-conserving surgery followed by radiotherapy is part of standard treatment for early-stage breast cancer. Hypoxia is common in cancer and may affect the benefit of radiotherapy. Cells adapt to hypoxic stress largely via the transcriptional activity of hypoxia-inducible factor (HIF)-1α. Here, we aim to determine whether tumour HIF-1α-positivity and hypoxic gene-expression signatures associated with the benefit of radiotherapy, and outcome. METHODS: Tumour HIF-1α-status and expression of hypoxic gene signatures were retrospectively analysed in a clinical trial where 1178 women with primary T1-2N0M0 breast cancer were randomised to receive postoperative radiotherapy or not and followed 15 years for recurrence and 20 years for breast cancer death. RESULTS: The benefit from radiotherapy was similar in patients with HIF-1α-positive and -negative primary tumours. Both ipsilateral and any breast cancer recurrence were more frequent in women with HIF-1α-positive primary tumours (hazard ratio, HR0-5 yrs1.9 [1.3-2.9], p = 0.003 and HR0-5 yrs = 2.0 [1.5-2.8], p < 0.0001). Tumour HIF-1α-positivity is also associated with increased breast cancer death (HR0-10 years 1.9 [1.2-2.9], p = 0.004). Ten of the 11 investigated hypoxic gene signatures correlated positively to HIF-1α-positivity, and 5 to increased rate/risk of recurrence. CONCLUSIONS: The benefit of postoperative radiotherapy persisted in patients with hypoxic primary tumours. Patients with hypoxic primary breast tumours had an increased risk of recurrence and breast cancer death.
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Neoplasias de la Mama , Mastectomía Segmentaria , Neoplasias de la Mama/genética , Neoplasias de la Mama/radioterapia , Neoplasias de la Mama/cirugía , Femenino , Estudios de Seguimiento , Humanos , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Recurrencia Local de Neoplasia/radioterapia , Pronóstico , Estudios RetrospectivosRESUMEN
BACKGROUND: Adjuvant endocrine treatment improves survival after estrogen receptor (ER) positive breast cancer. Recurrences occur, and most patients with metastatic breast cancer develop treatment resistance and incurable disease. An influential factor in relation to endocrine treatment resistance is tumor hypoxia and the hypoxia inducible transcription factors (HIFs). Poor perfusion makes tumors hypoxic and induces the HIFs, which promote cell survival. We previously showed that hypoxic breast cancer cells are tamoxifen-resistant, and that HIF-inhibition restored tamoxifen-sensitivity. We found that HIF-induced tamoxifen-resistance involve cross-talk with epithelial growth factor receptor (EGFR), which itself is linked to tamoxifen resistance. Contralateral breast cancer (CBC), i.e. development of a second breast cancer in the contralateral breast despite adjuvant tamoxifen treatment is in essence a human in vivo-model for tamoxifen-resistance that we explore here to find molecular pathways of tamoxifen-resistance. METHODS: We constructed a tissue-microarray including tumor-tissue from a large well-defined cohort of CBC-patients, a proportion of which got their second breast cancer despite ongoing adjuvant therapy. Using immunohistochemistry >500 patients were evaluable for HIF-1α and EGFR in both tumors, and correlations to treatment, patient outcome, prognostic and predictive factors were analyzed. RESULTS: We found an increased proportion of HIF-1α-positive tumors in tamoxifen-resistant (CBC during adjuvant tamoxifen) compared to naïve tumors (CBC without prior tamoxifen). Tumor HIF-1α-positivity correlated to increased breast cancer mortality, and negative prognostic factors including low age at diagnosis and ER-negativity. There was a covariance of HIF-1α- and EGFR-expression and also EGFR-expression correlated to poor prognosis. CONCLUSIONS: The increased percentage of HIF-1α-positive tumors formed during adjuvant tamoxifen suggests a role for HIF-1α in escaping tamoxifen's restraining effects on breast cancer. Implicating a potential benefit of HIF-inhibitors in targeting breast cancers resistant to endocrine therapy.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Tamoxifeno/uso terapéutico , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Persona de Mediana Edad , PronósticoRESUMEN
Tumor hypoxia correlates to aggressive disease, and while this is explained by a variety of factors, one clue to understand this phenomena was the finding that hypoxia induces a de-differentiated, stem cell-like phenotype in neuroblastoma and breast tumor cells. The hypoxia inducible transcription factors (HIFs) are regulated at the translational level by fluctuating oxygen concentrations, but emerging data reveal that both HIF-1α and HIF-2α expression can be induced by aberrantly activated growth factor signaling independently of oxygen levels. Furthermore, HIF-2α is regulated by hypoxia also at the transcriptional level in neuroblastoma and glioma cells. In cultured tumor cells, HIF-2α is stabilized at physiological oxygen concentrations followed by induced expression of classical hypoxia-driven genes, resulting in a pseudohypoxic phenotype. In addition, in neuroblastoma and glioma specimens, a small subset of HIF-2α positive, HIF-1α negative, tumor cells is found adjacent to blood vessels, i.e. in areas with presumably adequate oxygenation. These tumor niches are thus pseudohypoxic, and the HIF-2α expressing cells present immature features. We have postulated that this niche in neuroblastomas encompass the tumor stem cells. Oncogenes or tumor suppressor genes associated with pseudohypoxia are frequently mutated or deleted in the germline, implicating that the pseudohypoxic phenotype indeed is tumorigenic. In summary, the hypoxic and pseudohypoxic phenotypes of solid tumors are attractive therapeutic targets.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Hipoxia de la Célula/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Hipoxia/metabolismo , Neuroblastoma/metabolismo , Animales , HumanosRESUMEN
The majority of breast cancers express estrogen receptor α (ERα), and most patients with ERα-positive breast cancer benefit from antiestrogen therapy. The ERα-modulator tamoxifen and ERα-downregulator fulvestrant are commonly employed antiestrogens. Antiestrogen resistance remains a clinical challenge, with few effective treatments available for patients with antiestrogen-resistant breast cancer. Hypoxia, which is intrinsic to most tumors, promotes aggressive disease, with the hypoxia-inducible transcription factors HIF1 and HIF2 regulating cellular responses to hypoxia. Here, we show that the ERα-expressing breast cancer cells MCF-7, CAMA-1, and T47D are less sensitive to antiestrogens when hypoxic. Furthermore, protein and mRNA levels of HIF2α/HIF2A were increased in a panel of antiestrogen-resistant cells, and antiestrogen-exposure further increased HIF2α expression. Ectopic expression of HIF2α in MCF-7 cells significantly decreased sensitivity to antiestrogens, further implicating HIF2α in antiestrogen resistance. EGFR is known to contribute to antiestrogen resistance: we further show that HIF2α drives hypoxic induction of EGFR and that EGFR induces HIF2α expression. Downregulation or inhibition of EGFR led to decreased HIF2α levels. This positive and bilateral HIF2-EGFR regulatory crosstalk promotes antiestrogen resistance and, where intrinsic hypoxic resistance exists, therapy itself may exacerbate the problem. Finally, inhibition of HIFs by FM19G11 restores antiestrogen sensitivity in resistant cells. Targeting HIF2 may be useful for counteracting antiestrogen resistance in the clinic.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/fisiología , Receptores ErbB/metabolismo , Moduladores de los Receptores de Estrógeno/farmacología , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Femenino , Humanos , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiologíaRESUMEN
Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.
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Neoplasias de la Mama/patología , Neoplasias Pulmonares/secundario , Ganglios Linfáticos/patología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/fisiología , Células del Estroma/patología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ganglios Linfáticos/metabolismo , Metástasis Linfática , Ratones , Ratones Transgénicos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Células del Estroma/metabolismoRESUMEN
The development and functional cycle of the mammary gland involves a number of processes that are caricatured by breast cancer cells during invasion and metastasis. Expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 has been associated with metastatic, poor prognosis, and high-grade breast cancers. Since hypoxia affects normal epithelial differentiation, we hypothesise that HIFs are important for normal breast epithelial development and regeneration as well as cancer initiation and progression. Here, we investigated the expression of the oxygen-sensitive HIF-alpha subunits during mouse mammary gland development, lactation, and involution. In breast epithelial cells, HIF-1α was expressed during early development, prior to cell polarisation. In contrast, expression of HIF-2α occurred later and was restricted to a subpopulation of luminal epithelial cells in the lactating gland. Mammary gland involution is a developmental stage that involves extensive tissue remodelling with cell death but survival of tissue stem/progenitor cells. At this stage, HIF-2α, but little HIF-1α, was expressed in CK14-positive epithelial cells. The temporal but differential expression of the HIF-alpha subunits during the mammary gland life cycle indicates that their expression is controlled by additional factors to hypoxia. Further functional studies of the roles of these proteins in the mammary gland and breast cancer are warranted.
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Tejido Adiposo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Epiteliales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Glándulas Mamarias Animales/metabolismo , Animales , Hipoxia de la Célula , Células Cultivadas , Colágeno Tipo IV/metabolismo , Células Epiteliales/citología , Femenino , Inmunohistoquímica , Queratina-14/metabolismo , Lactancia , Células MCF-7 , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) is a protein with anti-protease and antimicrobial properties that is constitutively secreted from the airway epithelium. The importance of maintaining a balance between proteases and anti-proteases, and robust innate defence mechanisms in the airways, is exemplified by inflammatory lung conditions such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Both conditions present with a high protease burden in the airways which leads to tissue destruction. These patients also have an impaired innate immune system in the lungs with bacterial colonization and frequent airway infections. Moreover, both diseases are associated with airway hypoxia due to inflammation and mucus plugs. The aim of the present study was to investigate the role of hypoxia on SLPI production from the airway epithelium. METHODS: Primary human bronchial epithelial cells were grown in sub-immersed cultures or as differentiated epithelium in air liquid interface cultures. Cells were incubated at 21% O2 (normoxia) or 1% O2 (hypoxia), and the release of SLPI was analysed with ELISA. RT-PCR was used to study the expression of SLPI and transforming growth factor ß1 (TGF-ß1). RESULTS: Hypoxia decreased the constitutive production of SLPI by bronchial epithelial cells. The multifunctional cytokine TGF-ß1, which is known to affect SLPI expression, showed increased expression in hypoxic bronchial epithelial cells. When bronchial epithelial cells were exposed to exogenous TGF-ß1 during normoxia, the SLPI production was down-regulated. Addition of TGF-ß1-neutralizing antibodies partially restored SLPI production during hypoxia, showing that TGF-ß1 is an important regulator of SLPI during hypoxic conditions. CONCLUSIONS: The mechanism described here adds to our knowledge of the pathogenesis of severe pulmonary diseases associated with hypoxia, e.g. COPD and CF. The hypoxic down-regulation of SLPI may help explain the protease/anti-protease imbalance associated with these conditions and vulnerability to airway infections. Furthermore, it provides an interesting target for the treatment and prevention of exacerbation in these patients.
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Células Epiteliales/metabolismo , Hipoxia/genética , ARN Mensajero/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Factor de Crecimiento Transformador beta1/genética , Bronquios/citología , Bronquios/metabolismo , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Hipoxia/metabolismo , Elastasa de Leucocito/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα(+)) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.
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Proliferación Celular/efectos de los fármacos , Eritropoyetina/farmacología , Receptor alfa de Estrógeno/metabolismo , Receptores de Eritropoyetina/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Eritropoyetina/genética , Receptor alfa de Estrógeno/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Microscopía Confocal , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Receptores de Eritropoyetina/genética , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
INTRODUCTION: Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis. METHODS: Normal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot. RESULTS: In large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state.
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Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/patología , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Humanas/patología , Acetilación , Células Acinares/patología , Antígenos de Diferenciación/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Intraductal no Infiltrante/metabolismo , Moléculas de Adhesión Celular/metabolismo , Muerte Celular , Hipoxia de la Célula , Línea Celular , Polaridad Celular , Proliferación Celular , Células Epiteliales/patología , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal , Matriz Extracelular/fisiología , Femenino , Expresión Génica , Regulación de la Expresión Génica , Glucolípidos/metabolismo , Glicoproteínas/metabolismo , Histonas/metabolismo , Humanos , Proteína 1 Inhibidora de la Diferenciación/genética , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Integrina alfa6/metabolismo , Gotas Lipídicas , Fenotipo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , KalininaRESUMEN
The differentiation stage of tumors is a central aspect in the histopathological classification of solid malignancies. The differentiation stage is strongly associated with tumor behavior, and generally an immature tumor is more aggressive than the more differentiated counterpart. While this is common knowledge in surgical pathology, the contribution of differentiation-related gene expression and functions to tumor behavior is often overlooked in the experimental, tumor biological setting. The mechanisms by which tumor cell differentiation stages are perturbed or affected are poorly explored but have recently come into focus with the introduction.of the tumor stem cell concept. While developmental biologists view the differentiation as a unidirectional event, pathologists and tumor biologists have introduced the concept of dedifferentiation to explain phenotypic changes occurring in solid tumors. In this review we discuss the impact of the tumor cell differentiation stage as used in surgical pathology. We further discuss knowledge gained from exploring the molecular basis of the differentiation and dedifferentiation processes in neuroblastoma and breast cancer, two tumor forms where the tumor cell differentiation concept is used in the clinical diagnostic work and where the tumor stem cell theory has been applied.
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Diferenciación Celular , Neoplasias/patología , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal , Femenino , Humanos , Neuroblastoma/patologíaRESUMEN
Several protein kinase C (PKC) isoforms have been shown to influence different cellular processes that may contribute to the malignancy of breast cancer cells. To obtain insight into mechanisms mediating the PKC effects, global gene expression was analyzed in MDA-MB-231 breast cancer cells in which PKCα, PKCδ or PKCε had been down-regulated with siRNA. Gene set enrichment analyses revealed that hypoxia-induced genes were enriched among genes that increased in PKCα-down-regulated cells. The STC1 mRNA, encoding stanniocalcin 1, was particularly up-regulated following depletion of PKCα and was also induced by hypoxia. Both hypoxia and PKCα down-regulation also led to increased STC1 protein levels. The results demonstrate that PKCα suppresses the expression of STC1 in breast cancer cells.
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Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Glicoproteínas/biosíntesis , Proteína Quinasa C-alfa/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Glicoproteínas/genética , Humanos , Análisis por Micromatrices , Proteína Quinasa C-alfa/genética , ARN Mensajero/análisis , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND: Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficient mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficient mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murine inhibitory mU1 antibody provides a new and highly versatile tool to interfere with uPA-activity in vivo in mouse models of disease.
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Anticuerpos Monoclonales/administración & dosificación , Fibrinolisina/metabolismo , Piel/fisiopatología , Activador de Tejido Plasminógeno/deficiencia , Activador de Plasminógeno de Tipo Uroquinasa/inmunología , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pruebas de Neutralización , Piel/efectos de los fármacos , Piel/lesiones , Activador de Tejido Plasminógeno/genética , Heridas y Lesiones/metabolismo , Heridas y Lesiones/fisiopatologíaRESUMEN
Single-strand RNA-binding proteins (RBPs) are involved in many aspects of RNA metabolism and in the regulation of gene transcription. The RBP RBM3 was recently suggested to be a proto-oncogene in colorectal cancer; however, such a role has not been corroborated by previous studies in the colon or other tumor types, and the prognostic implications of tumor-specific RBM3 expression remain unclear. Mono-specific antibodies against RBM3 were generated. Antibody specificity was confirmed using siRNA gene silencing, western blotting and immunohistochemistry on a panel of breast cancer cell lines. Using tissue microarrays and IHC, RBM3 protein expression was examined in 48 normal tissues and in 20 common cancers. Additional analysis in two independent breast cancer cohorts (n=1016) with long-term follow-up was also carried out. RBM3 was upregulated in cancer compared to normal tissues. The nuclear expression of RBM3 in breast cancer was associated with low grade (P<0.001), small tumors (P<0.001), estrogen receptor (ER) positivity (P<0.001) and Ki-67 negativity (P<0.001) in both the breast cancer cohorts. An increased nuclear expression of RBM3 was associated with a prolonged overall and recurrence-free survival. The prognostic value was particularly pronounced in hormone receptor-positive tumors and remained significant in multivariate interaction analysis after controlling for tamoxifen treatment (HR: 0.49, 95% CI: 0.30-0.79, P=0.004). These data strongly indicate that nuclear RBM3 is an independent favorable prognostic factor in breast cancer, and seems to have a specific role in ER-positive tumors.
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Antineoplásicos Hormonales/uso terapéutico , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al ARN/metabolismo , Tamoxifeno/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Estudios de Cohortes , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Antígeno Ki-67/análisis , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Proto-Oncogenes Mas , Interferencia de ARN , Proteínas de Unión al ARN/inmunología , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores de Estrógenos/análisis , Medición de Riesgo , Factores de Tiempo , Análisis de Matrices Tisulares , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
INTRODUCTION: We have previously reported that tumour-specific expression of the rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR), in the mevalonate pathway is associated with more favourable tumour parameters in breast cancer. In the present study, we examined the prognostic value of HMG-CoAR expression in a large cohort of primary breast cancer patients with long-term follow up. METHODS: The expression of HMG-CoAR was assessed by immunohistochemistry on tissue microarrays with tumour specimens from 498 consecutive cases of breast cancer with a median follow-up of 128 months. Kaplan Meier analysis and Cox proportional hazards modelling were used to estimate the rate of recurrence-free survival (RFS) and breast cancer specific survival (BCSS). RESULTS: In line with our previous findings, tumour-specific HMG-CoAR expression was associated with low grade (p < 0.001), small size (p = 0.007), oestrogen receptor (ER) positive (p = 0.01), low Ki-67 (p = 0.02) tumours. Patients with tumours expressing HMG-CoAR had a significantly prolonged RFS, even when adjusted for established prognostic factors (relative risk [RR] = 0.60, 95% confidence interval [CI] 0.40 to 0.92; p = 0.02). In ER-negative tumours, however, there was a trend, that was not significantly significant, towards a shorter RFS in HMG-CoAR expressing tumours. CONCLUSIONS: HMG-CoAR expression is an independent predictor of a prolonged RFS in primary breast cancer. This may, however, not be true for ER-negative tumours. Further studies are needed to shed light on the value of HMG-CoAR expression as a surrogate marker of response to statin treatment, especially with respect to hormone receptor status.
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Neoplasias de la Mama/enzimología , Carcinoma/enzimología , Hidroximetilglutaril-CoA Reductasas/análisis , Proteínas de Neoplasias/análisis , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Hormonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/terapia , Carcinoma/mortalidad , Carcinoma/terapia , Quimioterapia Adyuvante , Estudios de Cohortes , Terapia Combinada , Supervivencia sin Enfermedad , Femenino , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Metástasis Linfática , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Receptores de Estrógenos/análisis , Tamoxifeno/uso terapéuticoRESUMEN
Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using gene-targeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.
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Fibrinólisis , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Anticuerpos/química , Anticuerpos Monoclonales/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Cinética , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Unión Proteica , Proteínas Recombinantes/química , Resonancia por Plasmón de SuperficieRESUMEN
Binding of urokinase plasminogen activator (uPA) to its cellular receptor, uPAR, potentiates plasminogen activation and localizes it to the cell surface. Focal plasminogen activation is involved in both normal and pathological tissue remodeling processes including cancer invasion. The interaction between uPA and uPAR therefore represents a potential target for anti-invasive cancer therapy. Inhibitors of the human uPA-uPAR interaction have no effect in the murine system. To enable in-vivo studies in murine cancer models we have now generated murine monoclonal antibodies (mAbs) against murine uPAR (muPAR) by immunizing uPAR-deficient mice with recombinant muPAR and screened for antibodies, which inhibit the muPA-muPAR interaction. Two of the twelve mAbs obtained, mR1 and mR2, interfered with the interaction between muPAR and the amino-terminal fragment of muPA (mATF) when analyzed by surface plasmon resonance. The epitope for mR1 is located on domain I of muPAR, while that of mR2 is on domains (II-III). In cell binding experiments using radiolabelled mATF, the maximal inhibition obtained with mR1 was 85% while that obtained with mR2 was 50%. The IC(50) value for mR1 was 0.67 nM compared to 0.14 nM for mATF. In an assay based on modified anthrax toxins, requiring cell-bound muPA activity for its cytotoxity, an approximately 50% rescue of the cells could be obtained by addition of mR1. Importantly, in-vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of uPA-activatable anthrax toxins.
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Anticuerpos Monoclonales/farmacología , Macrófagos/efectos de los fármacos , Plasminógeno/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Reacciones Antígeno-Anticuerpo , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/toxicidad , Sitios de Unión de Anticuerpos , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Mapeo Epitopo , Femenino , Humanos , Hibridomas/metabolismo , Inmunización , Radioisótopos de Yodo/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Factores de TiempoRESUMEN
Fast-growing solid tumors are usually insufficiently vascularized, leading to areas with necrosis and/or poorly oxygenated cells. Tumor cells adapt to acute hypoxic stress. Central to this adaptation are the hypoxia-inducible transcription factors (HIFs), which are degraded at normoxic but become stabilized at hypoxic conditions. Hypoxic (1% O2) neuroblastoma cells downregulate sympathetic nervous system marker genes, whereas neural crest cell markers are upregulated, suggesting that hypoxic tumor cells adopt a less mature phenotype, which in the clinical setting would translate to more aggressive tumors with increased metastatic potential. Here, we compared gene expression patterns in neuroblastoma cells grown at 1%, 5% (a physiologic oxygen level) and 21% O2. At 5% O2, cells developed a weak hypoxic phenotype and HIF-2 alpha, but not HIF-1 alpha, was acutely stabilized. At 1% O2, HIF-2 alpha protein remained present in long-term cultures, while HIF-1 alpha was present only transiently. The stability of the hypoxia-induced dedifferentiated phenotype in cells acutely reoxygenated at either 21% or 5% O2 persisted for at least 24 hr. Genes associated with a differentiated state, like NPY, ChrA and ChrB, were still downregulated and hypoxia-induced genes, like TH and Id2, remained upregulated. Thus, if these culture conditions are viewed as models for acute reoxygenation of metastasizing hypoxic tumor cells, our data suggest that an aggressive hypoxic phenotype persists for 24 hr or more, which might be long enough for the cells to be able to home to secondary sites, in part as a consequence of their immature hypoxic characteristics.
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Hipoxia de la Célula , Regulación Neoplásica de la Expresión Génica , Neuroblastoma/genética , Neuroblastoma/patología , Oxígeno/metabolismo , Adaptación Fisiológica , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Supervivencia Celular , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Metástasis de la Neoplasia , Fenotipo , Factores de Transcripción/biosíntesis , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Solid tumors are frequently necrotic and hypoxic due to poor vascularization. Tumor cells adapt to hypoxia by modulating their phenotype. Key players in this process are the hypoxia-inducible factors (HIF-1alpha to 3alpha). HIFs are also expressed during normal development; for example, HIF-2alpha is specifically expressed and appears to be involved in the development of the murine sympathetic nervous system (SNS). Here, we demonstrate that HIF-2alpha protein is selectively present in human fetal week 8.5 SNS paraganglia. Neuroblastoma is derived from SNS precursors. In a subset of neuroblastomas, a spontaneous neuronal to neuroendocrine differentiation occurs in areas adjacent to necrotic zones. As HIF-2alpha activity has been associated not only with hypoxic but also with hypoglycemic conditions, we have investigated putative effects of hypoxia, glucose depletion, and HIF-2alpha on the neuroblastoma phenotype. HIF-2alpha was detected in hypoxic and in well-oxygenized neuroblastoma cells and tissue, presumably reflecting their embryonic features. With regard to differentiation, hypoxic cells lost their neuronal/neuroendocrine features and gained marker gene expression associated with an immature, neural crest-like phenotype. Low glucose potentiated the effect of hypoxia. These findings suggest that poorly vascularized neuroblastomas become immature and maintain a more aggressive phenotype, which possibly could involve a sustained stabilization and activation of HIF-2alpha.
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Neuroblastoma/metabolismo , Paraganglios no Cromafines/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Hipoxia de la Célula , Línea Celular Tumoral , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glucosa/metabolismo , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Paraganglios no Cromafines/embriología , Sistema Nervioso Simpático/embriología , Sistema Nervioso Simpático/metabolismo , Factores de Transcripción/genéticaRESUMEN
ID (inhibitor of differentiation/DNA binding) proteins, frequently deregulated in advanced human malignancies, can participate in multiple fundamental traits of cancer, such as block of differentiation, increased proliferation, tissue invasiveness, and angiogenesis. We have previously demonstrated that hypoxia decreases expression of neuronal marker genes in neuroblastoma, but induces genes expressed in the neural crest, such as ID2. Because of its involvement in normal neural crest development and its ability to inhibit proneuronal bHLH proteins, the hypoxic induction of ID2 was of particular interest. Here we report fast induction kinetics of ID2 expression in hypoxic neuroblastoma cells. The up-regulation of ID2 was abolished by addition of actinomycin D, implicating a hypoxia-driven transcriptional mechanism. Analyzing the ID2 promoter revealed several potential binding sites for hypoxia-inducible factors. Subsequent electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated two functional HIF-1 binding sites within ID2 gene regulatory sequences located at -725 and -1893 relative to the transcriptional initiation point. In transfection assays, DNA constructs of the ID2 promoter, including the functional HIF-1 binding sites, induced luciferase reporter activity in a HIF-1-specific manner. These observations demonstrate that ID2 is actively engaged by hypoxia and represents a novel HIF-1 target. Hypoxia-induced ID2 expression could play a significant role in the previously observed dedifferentiation of hypoxic neuroblastoma cells, which in a clinical setting could lead to less mature and more aggressive tumors.