RESUMEN
Matched healthy and diseased tissues from breast cancer patients were analyzed by quantitative proteomics. By comparing proteomic profiles of fibroadenoma (benign tumors, three patients), DCIS (noninvasive cancer, three patients), and invasive ductal carcinoma (four patients), we identified protein alterations that correlated with breast cancer progression. Three 8-plex iTRAQ experiments generated an average of 826 protein identifications, of which 402 were common. After excluding those originating from blood, 59 proteins were significantly changed in tumor compared with normal tissues, with the majority associated with invasive carcinomas. Bioinformatics analysis identified relationships between proteins in this subset including roles in redox regulation, lipid transport, protein folding, and proteasomal degradation, with a substantial number increased in expression due to Myc oncogene activation. Three target proteins, cofilin-1 and p23 (increased in invasive carcinoma) and membrane copper amine oxidase 3 (decreased in invasive carcinoma), were subjected to further validation. All three were observed in phenotype-specific breast cancer cell lines, normal (nontransformed) breast cell lines, and primary breast epithelial cells by Western blotting, but only cofilin-1 and p23 were detected by multiple reaction monitoring mass spectrometry analysis. All three proteins were detected by both analytical approaches in matched tissue biopsies emulating the response observed with proteomics analysis. Tissue microarray analysis (361 patients) indicated cofilin-1 staining positively correlating with tumor grade and p23 staining with ER positive status; both therefore merit further investigation as potential biomarkers.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Cofilina 1/genética , Fibroadenoma/genética , Regulación Neoplásica de la Expresión Génica , Adulto , Anciano , Amina Oxidasa (conteniendo Cobre)/genética , Amina Oxidasa (conteniendo Cobre)/aislamiento & purificación , Amina Oxidasa (conteniendo Cobre)/metabolismo , Transporte Biológico , Biomarcadores de Tumor/aislamiento & purificación , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Estudios de Casos y Controles , Cofilina 1/aislamiento & purificación , Cofilina 1/metabolismo , Femenino , Fibroadenoma/diagnóstico , Fibroadenoma/metabolismo , Fibroadenoma/patología , Perfilación de la Expresión Génica , Humanos , Metabolismo de los Lípidos , Persona de Mediana Edad , Estadificación de Neoplasias , Oxidación-Reducción , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Proteolisis , Proteómica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Análisis de Matrices Tisulares , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
Cofilin1 is an actin-binding protein that plays a critical role in the regulation of actin cytoskeleton and consequently affects various physiological processes. In this study, the human Cofilin1 cDNA was cloned into the expression vector pET-28a(+) with a 6 × His tag and expressed as soluble protein in Escherichia coli BL21(DE3). Approximately 78 mg of Cofilin1, which showed high activity as determined by native PAGE, could be purified from each liter of LB medium by His-tag affinity chromatography and gel filtration. Further, high-titer IgG against Cofilin1 was positively detected after immunization in rabbits and the polyclonal antibodies were purified and identified. Together, this report provides the first protocol to efficiently obtain human Cofilin1 with high biological activity and immunogenicity using E. coli BL21 (DE3) expression system.