RESUMEN
Tumor-associated macrophages (TAM) are implicated in breast cancer metastasis, but relatively little is known about the underlying genes and pathways that are involved. The transcription factor Ets2 is a direct target of signaling pathways involved in regulating macrophage functions during inflammation. We conditionally deleted Ets in TAMs to determine its function at this level on mouse mammary tumor growth and metastasis. Ets2 deletion in TAMs decreased the frequency and size of lung metastases in three different mouse models of breast cancer metastasis. Expression profiling and chromatin immunoprecipitation assays in isolated TAMs established that Ets2 repressed a gene program that included several well-characterized inhibitors of angiogenesis. Consistent with these results, Ets2 ablation in TAMs led to decreased angiogenesis and decreased growth of tumors. An Ets2-TAM expression signature consisting of 133 genes was identified within human breast cancer expression data which could retrospectively predict overall survival of patients with breast cancer in two independent data sets. In summary, we identified Ets2 as a central driver of a transcriptional program in TAMs that acts to promote lung metastasis of breast tumors.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Macrófagos/metabolismo , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Proteína Proto-Oncogénica c-ets-2/fisiología , Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/metabolismo , Animales , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/mortalidad , Carcinoma/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Macrófagos/patología , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/mortalidad , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Proto-Oncogénica c-ets-2/genética , Estudios Retrospectivos , Células Tumorales CultivadasRESUMEN
The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Mama/efectos de los fármacos , Mama/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Receptores de Superficie Celular/metabolismo , Tretinoina/farmacología , Proteína de Unión a Vitamina D/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Mama/citología , Diferenciación Celular , Línea Celular , Cartilla de ADN/genética , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Proteínas Supresoras de Tumor , Proteína de Unión a Vitamina D/genéticaRESUMEN
The major circulating form of vitamin D is 25-hydroxycholecalciferol [25(OH)D3], which is delivered to target tissues in complex with the serum vitamin D binding protein (DBP). We recently observed that mammary cells can metabolize 25(OH)D3 to 1,25-dihydroxycholecalciferol [1,25(OH)(2)D3], the vitamin D receptor (VDR) ligand, and the objective of our study was to elucidate the mechanisms by which the 25(OH)D3-DBP complex is internalized by mammary cells prior to metabolism. Using fluorescent microscopy and temperature-shift techniques, we found that T-47D breast cancer cells rapidly internalize DBP via endocytosis, which is blunted by receptor-associated protein, a specific inhibitor of megalin-mediated endocytosis. Endocytosis of DBP was associated with activation of VDR by 25(OH)D3 but not 1,25(OH)(2)D3 (as measured by induction of the VDR target gene, CYP24). We also found that megalin and its endocytic partner, cubilin, are coexpressed in normal murine mammary tissue, in nontransformed human mammary epithelial cell lines, and in some established human breast cancer cell lines. To our knowledge, our studies are the first to demonstrate that mammary-derived cells express megalin and cubilin, which contribute to the endocytic uptake of 25(OH)D3-DBP and activation of the VDR pathway.
