RESUMEN
Stat3 is a signaling node for multiple oncogenic pathways and is therefore frequently active in breast cancer. As experimental and clinical evidence reveals that progestins are key players in controlling mammary gland tumorigenesis, we studied Stat3 participation in this event. We have previously shown that progestins induce Stat3Tyr705 phosphorylation and its transcriptional activation in breast cancer cells. In this study, we demonstrate that progestins also induce Stat3 phosphorylation at Ser727 residue, which occurs via activation of c-Src/p42/p44 MAPK pathways in murine progestin-dependent C4HD cells and in T-47D cells. Expression of a Stat3S727A vector, which carries a serine-to-alanine substitution at codon 727, shows that Stat3Ser727 phosphorylation is required for full transcriptional activation of cyclin D1 gene expression by progestins and for in vivo Stat3 recruitment on cyclin D1 promoter. Transfection of Stat3S727A in murine and human breast cancer cells abolished progestin-induced in vitro and in vivo growth. Moreover, we found a positive correlation between progesterone receptor expression and nuclear localization of Stat3Ser727 phosphorylation in breast cancer biopsies. These data highlight Stat3 phosphorylation in Ser727 residue as a nongenomic action by progestins, necessary to promote breast cancer growth.
Asunto(s)
Acetato de Medroxiprogesterona/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Proliferación Celular , Ciclina D1/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Fosforilación , Factor de Transcripción STAT3/genéticaRESUMEN
Aberrant Stat3 activation and signaling contribute to malignant transformation by promoting cell cycle progression, inhibiting apoptosis, and mediating tumor immune evasion. Stat3 inhibition in tumor cells induces the expression of chemokines and proinflammatory cytokines, so we proposed to apply Stat3-inhibited breast cancer cells as a source of immunogens to induce an antitumor immune response. Studies were performed in two murine breast cancer models in which Stat3 is activated: progestin-dependent C4HD cells and 4T1 cells. We immunized BALB/c mice with irradiated cancer cells previously transfected with a dominant-negative Stat3 vector (Stat3Y705F) in either a prophylactic or a therapeutic manner. Prophylactic administration of breast cancer cells transfected with Stat3Y705F (Stat3Y705F-breast cancer cells) inhibited primary tumor growth compared with administration of empty vector-transfected cells in both models. In the 4T1 model, 50% of the challenged mice were tumor free, and the incidence of metastasis decreased by 90%. In vivo assays of C4HD tumors showed that the antitumor immune response involves the participation of CD4(+) T cells and cytotoxic NK cells. Therapeutic immunization with Stat3Y705F-breast cancer cells inhibited tumor growth, promoted tumor cell differentiation, and decreased metastasis. Furthermore, inhibition of Stat3 activation in breast cancer cells induced cellular senescence, contributing to their immunogenic phenotype. In this work, we provide preclinical proof of concept that ablating Stat3 signaling in breast cancer cells results in an effective immunotherapy against breast cancer growth and metastasis. Moreover, our findings showing that Stat3 inactivation results in induction of a cellular senescence program disclose a potential mechanism for immunotherapy research.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Senescencia Celular/inmunología , Marcación de Gen , Células Asesinas Naturales/inmunología , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/terapia , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Línea Celular Tumoral , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Marcación de Gen/métodos , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Cultivo Primario de Células , Factor de Transcripción STAT3RESUMEN
BACKGROUND: The biological relevance of nuclear ErbB-2/HER2 (NuclErbB-2) presence in breast tumors remains unexplored. In this study we assessed the clinical significance of ErbB-2 nuclear localization in primary invasive breast cancer. The reporting recommendations for tumor marker prognostic studies (REMARK) guidelines were used as reference. METHODS: Tissue microarrays from a cohort of 273 primary invasive breast carcinomas from women living in Chile, a Latin American country, were examined for membrane (MembErbB-2) and NuclErbB-2 expression by an immunofluorescence (IF) protocol we developed. ErbB-2 expression was also evaluated by immunohistochemistry (IHC) with a series of antibodies. Correlation between NuclErbB-2 and MembErbB-2, and between NuclErbB-2 and clinicopathological characteristics of tumors was studied. The prognostic value of NuclErbB-2 in overall survival (OS) was evaluated using Kaplan-Meier method, and Cox model was used to explore NuclErbB-2 as independent prognostic factor for OS. RESULTS: The IF protocol we developed showed significantly higher sensitivity for detection of NuclErbB-2 than IHC procedures, while its specificity and sensitivity to detect MembErbB-2 were comparable to those of IHC procedures. We found 33.6% NuclErbB-2 positivity, 14.2% MembErbB-2 overexpression by IF, and 13.0% MembErbB-2 prevalence by IHC in our cohort. We identified NuclErbB-2 positivity as a significant independent predictor of worse OS in patients with MembErbB-2 overexpression. NuclErbB-2 was also a biomarker of lower OS in tumors that overexpress MembErbB-2 and lack steroid hormone receptors. CONCLUSIONS: We revealed a novel role for NuclErbB-2 as an independent prognostic factor of poor clinical outcome in MembErbB-2-positive breast tumors. Our work indicates that patients presenting NuclErbB-2 may need new therapeutic strategies involving specific blockage of ErbB-2 nuclear migration.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Proteínas Nucleares/metabolismo , Receptor ErbB-2/metabolismo , Adulto , Anciano , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Carcinoma/química , Chile , Estudios de Cohortes , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Análisis por Micromatrices , Persona de Mediana Edad , Proteínas Nucleares/análisis , Pronóstico , Modelos de Riesgos Proporcionales , Receptor ErbB-2/análisisRESUMEN
Interactions between progesterone receptor (PR) and signal transducer and activator of transcription 3 (Stat3)-mediated signaling pathways have already been described. In the present study, we explored the capacity of Stat3 to functionally interact with progesterone receptor (PR) and modulate PR transcriptional activation in breast cancer cells. We found that the synthetic progestin medroxyprogesterone acetate (MPA) induced the association of a PR/Stat3 complex in which Stat3 acts as a coactivator of PR. We demonstrated that Stat3 activation is required for MPA modulation of the endogenous genes bcl-X and p21(CIP1) which are involved in MPA-induced cell cycle regulation. Stat3 activity as a coactivator of PR was observed in both the classical and nonclassical ligand activated-PR transcriptional mechanisms, since the effects described were identified in the bcl-X promoter which contains a progesterone responsive element and in the p21(CIP1) promoter which carries Sp1 binding sites where PR is recruited via the transcription factor Sp1. The data herein presented identifies a potential therapeutic intervention for PR-positive breast tumors consisting of targeting Stat3 function or PR/Stat3 interaction which will result in the inhibition of PR function.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Receptores de Progesterona/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Receptores de Progesterona/agonistas , Elementos de Respuesta , Activación Transcripcional , Regulación hacia Arriba , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Progesterone receptor (PR) and ErbB-2 bidirectional cross talk participates in breast cancer development. Here, we identified a new mechanism of the PR and ErbB-2 interaction involving the PR induction of ErbB-2 nuclear translocation and the assembly of a transcriptional complex in which ErbB-2 acts as a coactivator of Stat3. We also highlighted that the function of ErbB-2 as a Stat3 coactivator drives progestin-induced cyclin D1 promoter activation. Notably, PR is also recruited together with Stat3 and ErbB-2 to the cyclin D1 promoter, unraveling a new and unexpected nonclassical PR genomic mechanism. The assembly of the nuclear Stat3/ErbB-2 transcriptional complex plays a key role in the proliferation of breast tumors with functional PR and ErbB-2. Our findings reveal a novel therapeutic intervention for PR- and ErbB-2-positive breast tumors via the specific blockage of ErbB-2 nuclear translocation.
Asunto(s)
Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Neoplasias Mamarias Experimentales/etiología , Neoplasias Mamarias Experimentales/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Factor de Transcripción STAT3/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Técnicas de Silenciamiento del Gen , Genes bcl-1 , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Acetato de Medroxiprogesterona/toxicidad , Ratones , Ratones Endogámicos BALB C , Progestinas/toxicidad , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Receptores de Progesterona/antagonistas & inhibidores , Receptores de Progesterona/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/genética , Transducción de Señal , Transcripción Genética/efectos de los fármacosRESUMEN
Tumor necrosis factor alpha (TNFalpha) is a pleiotropic cytokine which, acting locally, induces tumor growth. Accumulating evidence, including our findings, showed that TNFalpha is mitogenic in breast cancer cells in vitro and in vivo. In the present study, we explored TNFalpha involvement on highly aggressive ErbB-2-overexpressing breast cancer cells. We found that TNFalpha induces ErbB-2 phosphorylation in mouse breast cancer C4HD cells and in the human breast cancer cell lines SK-BR-3 and BT-474. ErbB-2 phosphorylation at Tyr877 residue was mediated by TNFalpha-induced c-Src activation. Moreover, TNFalpha promoted ErbB-2/ErbB-3 heterocomplex formation, Akt activation and NF-kappaB transcriptional activation. Inhibition of ErbB-2 by addition of AG825, an epidermal growth factor receptor/ErbB-2-tyrosine kinase inhibitor, or knockdown of ErbB-2 by RNA interference strategy, blocked TNFalpha-induced NF-kappaB activation and proliferation. However, the humanized monoclonal antibody anti-ErbB-2 Herceptin could not inhibit TNFalpha ability to promote breast cancer growth. Interestingly, our work disclosed that TNFalpha is able to transactivate ErbB-2 and use it as an obligatory downstream signaling molecule in the generation of mitogenic signals. As TNFalpha has been shown to be present in the tumor microenvironment of a significant proportion of human infiltrating breast cancers, our findings would have clinical implication in ErbB-2-positive breast cancer treatment.
Asunto(s)
Neoplasias de la Mama/genética , Genes erbB-2 , FN-kappa B/metabolismo , Proteínas de Neoplasias/biosíntesis , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/fisiología , Activación Transcripcional , Factor de Necrosis Tumoral alfa/fisiología , Animales , Neoplasias de la Mama/patología , División Celular , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Dimerización , Femenino , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Fosforilación , Proteínas Quinasas/fisiología , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/farmacología , Receptor ErbB-2/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Cross talk between the steroid hormone receptors for estrogen and progesterone (PR) and the ErbB family of receptor tyrosine kinases appears to be a hallmark of breast cancer growth, but its underlying mechanism remains poorly explored. Here we have highlighted signal transducer and activator of transcription 3 (Stat3) as a key protein activated by heregulin (HRG), a ligand of the ErbB receptors, through co-opted, ligand-independent PR function as a signaling molecule. Stat3 activation was an absolute requirement in HRG-induced mammary tumor growth, and targeting Stat3 effectively inhibited growth of breast cancer cells with activated HRG/ErbB-2 and PR. Our findings unravel a novel potential therapeutic intervention in PR- and ErbB-2-positive breast tumors, involving the specific blockage of PR signaling activity.
Asunto(s)
Proliferación Celular , Neoplasias Mamarias Animales/patología , Neurregulina-1/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Femenino , Neoplasias Mamarias Animales/etiología , Ratones , Ratones Endogámicos BALB C , Transducción de SeñalRESUMEN
Tumor necrosis factor alpha (TNF alpha) enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. Here, we explored the intracellular signaling pathways triggered by TNF alpha, the participation of TNF alpha receptor (TNFR) 1 and TNFR2 and the molecular mechanism leading to breast cancer growth. We demonstrate that TNFalpha induced proliferation of C4HD murine mammary tumor cells and of T47D cells through the activation of p42/p44 MAPK, JNK, PI3-K/Akt pathways and nuclear factor-kappa B (NF-kappa B) transcriptional activation. A TNF alpha-specific mutein selectively binding to TNFR1 induced p42/p44 MAPK, JNK, Akt activation, NF-kappa B transcriptional activation and cell proliferation, just like wild-type TNF alpha, while a mutein selective for TNFR2 induced only p42/p44 MAPK activation. Interestingly, blockage of TNFR1 or TNFR2 with specific antibodies was enough to impair TNF alpha signaling and biological effect. Moreover, in vivo TNF alpha administration supported C4HD tumor growth. We also demonstrated, for the first time, that injection of a selective inhibitor of NF-kappa B activity, Bay 11-7082, resulted in regression of TNF alpha-promoted tumor. Bay 11-7082 blocked TNF alpha capacity to induce cell proliferation and up-regulation of cyclin D1 and of Bcl-xLin vivo and in vitro. Our results reveal evidence for TNF alpha as a breast tumor promoter, and provide novel data for a future therapeutic approach using TNF alpha antagonists and NF-kappa B pharmacological inhibitors in established breast cancer treatment.