title: Global phosphotyrosine proteomics identifies PKCδ as a marker of responsiveness to Src inhibition in colorectal cancer.
abstract: Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTPα). The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.
authors.affiliation: Vanderbilt University Institute of Imaging Science, Vanderbilt University, Nashville, Tennessee, United States of America ; Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, United States of America.
authors.forename: Eliot T
authors.initials: ET
authors.lastname: McKinley
authors.forename: Huiling
authors.initials: H
authors.lastname: Liu
authors.forename: W Hayes
authors.initials: WH
authors.lastname: McDonald
authors.forename: Weifeng
authors.initials: W
authors.lastname: Luo
authors.forename: Ping
authors.initials: P
authors.lastname: Zhao
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Coffey
authors.forename: Steven K
authors.initials: SK
authors.lastname: Hanks
authors.forename: H Charles
authors.initials: HC
authors.lastname: Manning
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor / Q000378:metabolism*; D018938:Caco-2 Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000378:metabolism*; D000069439:Dasatinib; D045325:HCT116 Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000378:metabolism; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D011743:Pyrimidines / Q000494:pharmacology; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5 / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C432044:CDCP1 protein, human; D015815:Cell Adhesion Molecules; D009363:Neoplasm Proteins; D047428:Protein Kinase Inhibitors; D020543:Proteome; D011743:Pyrimidines; D013844:Thiazoles; D019000:Phosphotyrosine; D019061:src-Family Kinases; D051745:Protein Kinase C-delta; D010744:Phosphoric Monoester Hydrolases; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D000069439:Dasatinib