Evaluation of a tyrosine kinase peptide microarray for tyrosine kinase inhibitor therapy selection in cancer.
Exp Mol Med
; 48(12): e279, 2016 12 16.
Article
in En
| MEDLINE
| ID: mdl-27980342
ABSTRACT
Personalized cancer medicine aims to accurately predict the response of individual patients to targeted therapies, including tyrosine kinase inhibitors (TKIs). Clinical implementation of this concept requires a robust selection tool. Here, using both cancer cell lines and tumor tissue from patients, we evaluated a high-throughput tyrosine kinase peptide substrate array to determine its readiness as a selection tool for TKI therapy. We found linearly increasing phosphorylation signal intensities of peptides representing kinase activity along the kinetic curve of the assay with 7.5-10 µg of lysate protein and up to 400 µM adenosine triphosphate (ATP). Basal kinase activity profiles were reproducible with intra- and inter-experiment coefficients of variation of <15% and <20%, respectively. Evaluation of 14 tumor cell lines and tissues showed similar consistently high phosphorylated peptides in their basal profiles. Incubation of four patient-derived tumor lysates with the TKIs dasatinib, sunitinib, sorafenib and erlotinib primarily caused inhibition of substrates that were highly phosphorylated in the basal profile analyses. Using recombinant Src and Axl kinase, relative substrate specificity was demonstrated for a subset of peptides, as their phosphorylation was reverted by co-incubation with a specific inhibitor. In conclusion, we demonstrated robust technical specifications of this high-throughput tyrosine kinase peptide microarray. These features required as little as 5-7 µg of protein per sample, facilitating clinical implementation as a TKI selection tool. However, currently available peptide substrates can benefit from an enhancement of the differential potential for complex samples such as tumor lysates. We propose that mass spectrometry-based phosphoproteomics may provide such an enhancement by identifying more discriminative peptides.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Protein-Tyrosine Kinases
/
Drug Screening Assays, Antitumor
/
Protein Kinase Inhibitors
/
Neoplasms
Limits:
Humans
Language:
En
Journal:
Exp Mol Med
Journal subject:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Year:
2016
Document type:
Article
Affiliation country:
Netherlands