RESUMEN
The incretin hormone glucagon-like peptide (GLP)-1 is secreted from intestinal L cells in response to food intake, and promotes insulin secretion and pancreatic ß-cell proliferation. Reduced GLP-1 levels are observed in obesity and type 2 diabetes mellitus (T2DM) and are associated with reduced insulin secretion and increased insulin resistance. GLP-1 mediates its activities through activation of a G-protein coupled receptor, which is expressed in the pancreas, as well as other tissues. Long-acting GLP-1 receptor (GLP-1R) agonists, such as exendin-4, are currently approved for the treatment of T2DM. As obesity and T2DM are associated with increased risk of breast cancer, we aimed to explore the effects of GLP-1 and exendin-4, on breast cancer cells. Treatment with GLP-1 or exendin-4 reduced viability and enhanced apoptosis of breast cancer cells but did not affect viability of nontumorigenic cells. Moreover, exendin-4 attenuated tumor formation by breast cancer cells in athymic mice. Treatment with either GLP-1 or exendin-4 elevated cAMP levels, activated the down-stream target CREB, and enhanced CRE promoter transcription, in breast cancer cells. Moreover, inhibition of exendin-4-induced adenylate cyclase activation restored cell viability, thus suggesting cAMP as a principle mediator of exendin-4 anti-tumorigenic activity. While the pancreatic form of the GLP-1R could not be detected in breast cancer cells, several lines of evidence indicated the existence of an alternative GLP-1R in mammary cells. Thus, internalization of GLP-1 into MCF-7 cells was evidenced, infection of MCF-7 cells with the pancreatic receptor enhanced proliferation, and treatment with exendin-(9-39), a GLP-1R antagonist, further increased cAMP levels. Our studies indicate the incretin hormone GLP-1 as a potent inducer of cAMP and an inhibitor of breast cancer cell proliferation. Reduced GLP-1 levels may, therefore, serve as a novel link between obesity, diabetes mellitus, and breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Ponzoñas/farmacología , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Exenatida , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptor del Péptido 1 Similar al Glucagón , Humanos , Ratones , Ratones Desnudos , Receptores de Glucagón/agonistas , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Elementos de Respuesta/efectos de los fármacos , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos , Transfección , Regulación hacia Arriba , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.