RESUMEN
BACKGROUND: The development and progression of estrogen receptor alpha positive (ERα+) breast cancer has been linked epidemiologically to prolactin. However, activation of the canonical mediator of prolactin, STAT5, is associated with more differentiated cancers and better prognoses. We have reported that density/stiffness of the extracellular matrix potently modulates the repertoire of prolactin signals in human ERα + breast cancer cells in vitro: stiff matrices shift the balance from the Janus kinase (JAK)2/STAT5 cascade toward pro-tumor progressive extracellular regulated kinase (ERK)1/2 signals, driving invasion. However, the consequences for behavior of ERα + cancers in vivo are not known. METHODS: In order to investigate the importance of matrix density/stiffness in progression of ERα + cancers, we examined tumor development and progression following orthotopic transplantation of two clonal green fluorescent protein (GFP) + ERα + tumor cell lines derived from prolactin-induced tumors to 8-week-old wild-type FVB/N (WT) or collagen-dense (col1a1 tm1Jae/+ ) female mice. The latter express a mutant non-cleavable allele of collagen 1a1 "knocked-in" to the col1a1 gene locus, permitting COL1A1 accumulation. We evaluated the effect of the collagen environment on tumor progression by examining circulating tumor cells and lung metastases, activated signaling pathways by immunohistochemistry analysis and immunoblotting, and collagen structure by second harmonic generation microscopy. RESULTS: ERα + primary tumors did not differ in growth rate, histologic type, ERα, or prolactin receptor (PRLR) expression between col1a1 tm1Jae/+ and WT recipients. However, the col1a1 tm1Jae/+ environment significantly increased circulating tumor cells and the number and size of lung metastases at end stage. Tumors in col1a1 tm1Jae/+ recipients displayed reduced STAT5 activation, and higher phosphorylation of ERK1/2 and AKT. Moreover, intratumoral collagen fibers in col1a1 tm1Jae/+ recipients were aligned with tumor projections into the adjacent fat pad, perpendicular to the bulk of the tumor, in contrast to the collagen fibers wrapped around the more uniformly expansive tumors in WT recipients. CONCLUSIONS: A collagen-dense extracellular matrix can potently interact with hormonal signals to drive metastasis of ERα + breast cancers.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Colágeno Tipo I/metabolismo , Receptor alfa de Estrógeno/metabolismo , Prolactina/metabolismo , Transducción de Señal , Animales , Biomarcadores , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Colágeno Tipo I/genética , Progresión de la Enfermedad , Matriz Extracelular/metabolismo , Femenino , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Células Neoplásicas Circulantes , Proteínas Proto-Oncogénicas c-akt , Factor de Transcripción STAT5/metabolismo , Carga TumoralRESUMEN
Prolactin (PRL) and estrogen cooperate in lobuloalveolar development of the mammary gland and jointly regulate gene expression in breast cancer cells in vitro. Canonical PRL signaling activates STAT5A/B, homologous proteins that have different target genes and functions. Although STAT5A/B are important for physiological mammary function and tumor pathophysiology, little is known about regulation of their expression, particularly of STAT5B, and the consequences for hormone action. In this study, we examined the effect of two estrogenic ligands, 17ß-estradiol (E2) and the clinical antiestrogen, ICI182,780 (ICI, fulvestrant) on expression of STAT5 isoforms and resulting crosstalk with PRL in normal and tumor murine mammary epithelial cell lines. In all cell lines, E2 and ICI significantly increased protein and corresponding nascent and mature transcripts for STAT5A and STAT5B, respectively. Transcriptional regulation of STAT5A and STAT5B by E2 and ICI, respectively, is associated with recruitment of estrogen receptor alpha and increased H3K27Ac at a common intronic enhancer 10 kb downstream of the Stat5a transcription start site. Further, E2 and ICI induced different transcripts associated with differentiation and tumor behavior. In tumor cells, E2 also significantly increased proliferation, invasion, and stem cell-like activity, whereas ICI had no effect. To evaluate the role of STAT5B in these responses, we reduced STAT5B expression using short hairpin (sh) RNA. shSTAT5B blocked ICI-induced transcripts associated with metastasis and the epithelial mesenchymal transition in both cell types. shSTAT5B also blocked E2-induced invasion of tumor epithelium without altering E2-induced transcripts. Together, these studies indicate that STAT5B mediates a subset of protumorigenic responses to both E2 and ICI, underscoring the need to understand regulation of its expression and suggesting exploration as a possible therapeutic target in breast cancer.
RESUMEN
Prolactin (PRL) modulates proliferation in the mammary gland and other tissues, in part through inducing transcription of cyclin D1, a key regulator of G(1) phase cell cycle progression. We showed previously that PRL, via Jak2, induces binding of Stat5 to a distal GAS site (GAS1) in the cyclin D1 promoter. However, full promoter activity requires additional regions, and in this paper we explored PRL-induced activity at sites other than GAS1. We defined a second PRL-responsive region spanning -254 to -180 that contains a second GAS site (GAS2) and an Oct-1 binding site. Although mutational analysis indicated independence from GAS2, proximal promoter activity remained Stat5-dependent, suggesting alternative mechanisms. EMSA showed that Oct-1 binds the -254 to -180 region and that PRL decreased Oct-1 binding, leading to increased PRL-responsiveness of the proximal cyclin D1 promoter in multiple cell lines. This suggests a role for Oct-1 in PRL-dependent control of cyclin D1 transcription.
Asunto(s)
Ciclina D1/biosíntesis , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Leche/metabolismo , Prolactina/farmacología , Regiones Promotoras Genéticas/fisiología , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Ciclina D1/genética , Regulación de la Expresión Génica/genética , Humanos , Janus Quinasa 2 , Mutación , Factor 1 de Transcripción de Unión a Octámeros , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT5 , Transducción de Señal/genéticaRESUMEN
PRL promotes cell growth and differentiation in the mammary gland, which has implications for breast cancer as well as normal development. Our data demonstrate that PRL significantly increases proliferation of mammary carcinoma cells. PRL also increases cyclin D1 levels 2-fold, which can be inhibited by actinomycin D, suggesting that transcriptional increases in cyclin D1 are important. Using a defined Chinese hamster ovary cell model system, we demonstrate that the activity of a cyclin D1 promoter-luciferase construct increases after PRL treatment. Furthermore, this increase in promoter activity is predominantly mediated by the Jak2/Stat5 signaling pathway. The cyclin D1 promoter contains two consensus sequences for PRL-induced Stat binding (GAS sites). Disruption of Stat binding to the distal GAS site destroys PRL-induced promoter activity, whereas disruption of the proximal site has no effect. We have shown by EMSA that PRL induces Stat5a and 5b to bind to the distal GAS site, and immunoprecipitation and subsequent Western analysis of nuclear extracts from PRL-treated cells indicate that Stat5a and 5b can interact as a heterodimer in this system. These data suggest that cyclin D1 may be a target gene for PRL in normal lobuloalveolar development, as well as in the development and/or progression of mammary cancer.
Asunto(s)
Neoplasias de la Mama/patología , Ciclina D1/genética , Proteínas de Unión al ADN/fisiología , Proteínas de la Leche , Prolactina/fisiología , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas , Transactivadores/fisiología , Secuencia de Aminoácidos , Animales , Células CHO , División Celular/efectos de los fármacos , Secuencia de Consenso , Cricetinae , Análisis Mutacional de ADN , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Janus Quinasa 2 , Datos de Secuencia Molecular , Factor de Transcripción STAT5 , Transducción de Señal , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Posttranslational modifications of prolactin (PRL), including phosphorylation, vary with physiologic state and alter biologic activity. In light of the growing evidence for a role for PRL in proliferation in mammary cancer, we examined the ability of a mimic of phosphorylated human PRL, S179D-PRL, to initiate signals to several pathways in mammary tumor cells alone and in combination with unmodified PRL. Unmodified PRL employed multiple pathways to increase cellular proliferation and cyclin D1 levels in PRL-deficient MCF-7 cells. S179D-PRL was a weak agonist compared with unmodified PRL with regard to cellular proliferation, cyclin D1 levels, and phosphorylation of signal transducer and activator of transcription 5 and ERKs. However, S179D-PRL was a potent antagonist of unmodified PRL to these endpoints. In contrast to the reduced levels of the long isoform of the PRL receptor observed in response to a 3-d incubation with unmodified PRL, S179D-PRL up-regulated expression of this isoform, 4-fold. These studies support the utility of this mutant as a PRL antagonist to proliferative signals in mammary epithelial cells, including a potential role in breast cancer therapeutics.