Abstract 4977: Dramatic differences in the extents and patterns of PKC delta modification among the different members of the NCI-60 cancer cell line panel

Abstract

Abstract Protein kinase Cs (PKCs) are validated targets for cancer therapy with many agents in clinical trials which affect their activity and/or expression. PKC delta is one of the most highly expressed PKC isoforms and typically functions biologically to promote apoptosis. Multiple phosphorylation sites, both on serine/threonine and on tyrosine, have been identified on PKC delta and various sites have been shown to be differentially important for the various biological activities of PKC delta. Unfortunately, phosphosite specific antibodies are only available for a few of these phosphorylation sites. Capillary isoelectric focusing on a nanoscale with immunoassay readout, using pan-reactive antibodies that detect all modified states, reveals signatures of the differently modified states of PKC delta in cells. In LNCaP cells, the phosphorylation pattern of PKC delta was modestly complex under basal conditions but it became highly complex after ligand treatment. This phosphorylation signature depended on the ligand and the subsequent subcellular localization of PKC delta. To assess the generality of the findings in the LNCaP cells, we have extended our analysis to assess the phosphorylation signatures of PKC delta in different cell lines of the NCI-60 cancer cell line panel. Under basal conditions, the various cell lines showed dramatic differences. Some of them (SR leukemia, HT-29 colon cancer, OVCAR-5 ovarian cancer, many lung cancer cell lines, T-47D breast cancer) showed a pattern similar to that of LNCaP cells. Many others (most renal cell lines, OVCAR-8 ovarian cancer, MDA-MB-468 breast cancer, SK-MEL5 melanoma) displayed a complex pattern of highly phosphorylated peaks in the PKC delta signature. The extents of PKC delta phosphorylation did not correlate with the sensitivity of the cell lines to growth inhibition by the PKC ligand mezerein. Current efforts with selected cell lines from the NCI-60 cell line panel are directed at 1) characterizing in detail the PKC delta signature using different phosphosite specific antibodies to deconvolute the modifications responsible for the multiplicity of the peaks, 2) exploring what factors influence the PKC delta signature, and 3) evaluating whether the basal PKC delta phosphorylation signature is a measure of overall DAG signaling or is specific to the PKC delta isoform. We conclude that capillary isoelectric focusing with immunoassay is a powerful tool for exploring the complexity of PKC delta activation/modification in different cells and for identifying those cancer types where dysregulation of the DAG signaling pathway is most prominent. While the current studies focus on PKC delta, this same methodology should be similarly informative for the many other proteins with an intermediate level of complexity in their post-translational modifications. Citation Format: Noemi Kedei, Jin-Qiu Chen, Michelle Herrmann, Tiffany Hu, Karina Chang, Peter M. Blumberg. Dramatic differences in the extents and patterns of PKC delta modification among the different members of the NCI-60 cancer cell line panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2015-4977