Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay.
Breast cancer subtype-specific molecular variations can dramatically affect patient responses to existing therapies. It is thought that differentially phosphorylated protein isoforms might be a useful prognostic biomarker of drug response in the clinic. However, the accurate detection and quantitative analysis of cancer-related protein isoforms and phospho-isoforms in tumors are limited by current technologies. Using a novel, fully automated nanocapillary electrophoresis immunoassay (NanoPro(TM) 1000) designed to separate protein molecules based on their isoelectric point, we developed a reliable and highly sensitive assay for the detection and quantitation of AKT isoforms and phosphoforms in breast cancer. This assay enabled the measurement of activated AKT1/2/3 in breast cancer cells using protein produced from as few as 56 cells. Importantly, we were able to assign an identity for the phosphorylated S473 phosphoform of AKT1, the major form of activated AKT involved in multiple cancers, including breast, and a current focus in clinical trials for targeted intervention. The ability of our AKT assay to detect and measure AKT phosphorylation from very low amounts of total protein will allow the accurate evaluation of patient response to drugs targeting activated PI3K-AKT using scarce clinical specimens. Moreover, the capacity of this assay to detect and measure all three AKT isoforms using one single pan-specific antibody enables the study of the multiple and variable roles that these isoforms play in AKT tumorigenesis.
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
Demetris C
DC
Iacovides
Aimee B
AB
Johnson
Nick
N
Wang
Shanta
S
Boddapati
Jim
J
Korkola
Joe W
JW
Gray
D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology*; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D000077341:Lapatinib; D053858:Metabolic Networks and Pathways; D046210:Microfluidic Analytical Techniques / Q000379:methods; D036103:Nanotechnology / Q000379:methods; D010069:Oxadiazoles / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D040901:Proteomics / Q000379:methods; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology
C528328:GSK690693; D007527:Isoenzymes; D010069:Oxadiazoles; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D000077341:Lapatinib; C494918:AKT1 protein, human; C494921:AKT2 protein, human; C491754:AKT3 protein, human; D051057:Proto-Oncogene Proteins c-akt