Identification of metastasis-associated proteins through protein analysis of metastatic MDA-MB-435 and metastasis-suppressed BRMS1 transfected-MDA-MB-435 cells.
BRMS1 (breast cancer metastasis suppressor 1) was recently identified as a novel breast cancer metastasis suppressor gene. To further characterize BRMS1-mediated metastasis suppression, we applied two-dimensional proteomic and mass spectrometry (LC-tandem MS and MALDI-TOF) analysis to identify proteins differentially expressed between highly metastatic MDA-MB-435 cells and metastasis-suppressed BRMS1-transfected MDA-MB-435 cells. Quadruplicate independent 2D gels were run and analyzed under identical conditions. Following in-gel trypsin digestion of seven differentially expressed proteins, amino acid sequence and mass profiles of the peptides were generated. Proteins were identified from the NCBI non-redundant database using the search program TurboSequest. Differential expression was confirmed for five proteins, including annexin I and alpha B-crystallin, by Northern blot analysis and immunostaining. Furthermore, we showed that both proteins were expressed in vivo in lungs containing metastasized MDA-MB-435 cells but not expressed in normal lung tissue of athymic mice. Our results suggest that annexin I and alpha B-crystallin are important cellular proteins that are down regulated through BRMS1 mediated metastasis suppression.
Cicek
Muzaffer
M
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Samant
Rajeev S
RS
Kinter
Michael
M
Welch
Danny R
DR
Casey
Graham
G
D000595:Amino Acid Sequence; D000818:Animals; D017305:Annexin A1 / Q000096:biosynthesis / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D044584:Carcinoma, Ductal / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000556:secondary*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000556:secondary; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D012097:Repressor Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013382:Subtraction Technique; D014162:Transfection; D014183:Transplantation, Heterologous; D038203:alpha-Crystallin B Chain / Q000096:biosynthesis / Q000235:genetics
D017305:Annexin A1; C410024:BRMS1 protein, human; D009363:Neoplasm Proteins; D012097:Repressor Proteins; D038203:alpha-Crystallin B Chain