Global Phosphotyrosine Proteomics Identifies PKCδ as a Marker of Responsiveness to Src Inhibition in Colorectal Cancer

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.

蛋白激酶抑制的灵敏且特异性生物标志物，可通过将早期分子应答与靶点抑制相关联，助力加速肿瘤学领域的药物开发研究。本研究采用无偏倚鸟枪法磷酸酪氨酸（pY）蛋白质组学，在结直肠癌（CRC）临床前模型中筛选达沙替尼（dasatinib，一种小分子Src选择性抑制剂）应答的新型生物标志物。我们先通过无偏倚质谱鸟枪法pY蛋白质组学，解析了体外培养的HCT-116结肠癌细胞的pY蛋白质组，随后将该分析拓展至HCT-116异种移植瘤，以在体内鉴定达沙替尼应答相关的pY生物标志物。异种移植瘤中达沙替尼应答的主要pY位点包括δ型蛋白激酶C（PKCδ）、含CUB结构域蛋白1（CDCP1）、II型含SH2结构域肌醇5-磷酸酶（SHIP2）以及受体型蛋白酪氨酸磷酸酶α（RPTPα）上的相关位点。借助磷酸化特异性抗体开展免疫组织化学与免疫印迹实验，进一步验证了PKCδ的pY313位点可作为HCT-116异种移植瘤中达沙替尼介导的Src抑制的相关生物标志物。在一组人类结直肠癌细胞系的细胞球实验中，PKCδ pY313水平的下调还与达沙替尼介导的Src抑制及肿瘤生长减弱呈显著相关。本研究表明，PKCδ pY313可作为结直肠癌乃至其他实体瘤中Src抑制的有效检测指标，或可反映部分结直肠癌患者对达沙替尼的应答情况。