Abstract 3889: An aptamer-based multiplexed proteomic technology reveals differential PKC ligand responses
Authors: Chen Jinqiu , Kedei Noemi , Goldstein David J. , Malik Mariam Q. , Blumberg Peter M. 
Published: July 2016

Bryostatin 1 (Bryo 1) is in clinical trials for cancer and Alzheimer's disease. Whereas Bryo 1, like the phorbol esters, binds to and activates protein kinase C (PKC), it paradoxically antagonizes many but not all phorbol ester responses. The mechanistic basis for this differential response is still unknown.

In the LNCaP human prostate cancer cell line, the typical phorbol ester PMA causes growth inhibition whereas Bryo 1 does not. Previously, we showed that PMA induces dramatic changes in gene transcription in the LNCaP cells as determined by gene expression microarray analysis. The effects of Bryo 1 on gene expression were initially similar at 1 hr but were variably more transient, as seen at 6 hr, depending on the specific gene. We think that this transiency of action is a critical mechanistic feature of Bryo 1.

The current study extends to the comparison of the effects of Bryo 1 and PMA on the level of global protein expression. Using a new generation of protein-capture SOMAmer (Slow Off-rate Modified Aptamer) reagents, the SOMAscan (Somalogic, Boulder, CO) proteomic assay simultaneously measures over one thousand analytes within a minute amount of protein sample. SOMAmer reagents are chemically modified single stranded DNA-based protein affinity reagents that recognize specific conformational epitopes of native 3D proteins with good specificity and dynamic range. The technology is emerging as a highly sensitive, multiplexed and quantitative proteomic tool for biomarker discovery and validation. 1,129 targets (covering major gene families including receptors, kinases, growth factors, and a diverse collection of secreted, intracellular and extracellular proteins or domains) were evaluated in both total cell lysate and nuclear extracts of LNCaP cells treated with DMSO, PMA and Bryo 1 for 60 minute and 6 hours. Compared to the DMSO control, the lysates of cells treated for 60 minute with either PMA and Bryo 1 had over three hundred targets that showed significant changes (p&lt;0.01). While a similar scale of signaling response was observed in the 6 hour PMA treated total cell lysate, less than 50 targets differed from the DMSO control in the 6 hour Bryo 1 treated samples (p&lt;0.01). Similar results were obtained for the nuclear extracts. Good assay reproducibility was observed within replicates. While the protein analysis confirmed the transient nature of Bryo 1 action, comparison of the SOMAscan protein expression data with the mRNA transcript analysis revealed substantial differences, indicating the importance of monitoring signaling response at both the transcriptomic and proteomic levels. It further identified a rich set of protein targets involved in the differential PKC signaling by different ligands. On-going studies seek to characterize the basis for the transient signaling response following Bryo 1 treatment.

Citation Format: Jinqiu Chen, Noemi Kedei, David J. Goldstein, Mariam Q. Malik, Peter M. Blumberg. An aptamer-based multiplexed proteomic technology reveals differential PKC ligand responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3889.