Abstract 5315: Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation

Abstract

Abstract Bryostatin 1 (bryo 1) is an anticancer agent currently in multiple clinical trials with a unique mechanism of action. Whereas bryo 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes many but not all phorbol ester responses. In LNCaP prostate cancer cells the typical phorbol ester PMA induces TNFalpha secretion and apoptosis while bryo 1 fails to do so and antagonizes the PMA effect when used in combination. The mechanisms responsible for the unique effect of bryo 1 are still unknown. We have shown previously, using transcription factor analysis of microarray data from LNCaP cells, that multiple transcription factor families are involved in the dramatic transcriptional response to PMA and bryo 1 (3131 genes for PMA and 1569 genes for bryo 1). Further, analysis of 45 signaling pathways using reporter assays identified significant changes in 7 pathways induced both by PMA and bryo 1 at 6 hours. To further investigate the altered pathways, the novel Simple Western capillary immunoassay system (ProteinSimple) was used to analyze protein level changes following PKC activation. Simple Western system performs automated western analysis in capillaries using nanograms of lysate and provides digital quantitation with good sensitivity and excellent reproducibility. The system runs at a through-put of 96 sample / target combinations in a single overnight run. We analyzed 35 signals in total lysates (for detection of changes in protein levels and phosphorylation) and in nuclear extracts (for detection of translocation) from LNCaP cells treated for 60 min and 6 hrs. Changes downstream of PKC activation were measured, including PKC/PKD1 phosphorylation, activation of multiple members of the MEK/ERK pathway and levels of transcription factors of the NFKB, AP1, Stat and EGR families. Simple Western results correlated very well with conventional western analysis. The analysis validated the changes described previously using other methods on activation of MEK/ERK pathway and the changes in PKC phosphorylation, translocation and degradation. Bryo1 is different from PMA in failing to induce phosphorylation of PKCdelta at Tyr311 and the translocation of PKCalpha, delta, epsilon, and PKD1 to the nuclear fraction. Bryo1 is similar to PMA in inducing many of the early responses detected at 60 min (pPKCdeltaSer299, pMEK, pERK1/2, pPKD1 in total lysates, decrease in STAT3 levels etc.) but it fails to sustain the induced responses or to induce the late responses detected at 6 hours (pNFκBp65, increase in Fra1 and FosB levels, degradation of cMyc, translocation of cRel and RelB etc.). Using Simple Western system, we developed an approach for precise and accurate measurement of differential PMA and bryo 1 signaling in PKC activation. Further investigation needs to be performed to identify the mechanism/s responsible for the early termination of bryo 1 induced responses. Citation Format: Jin-Qiu Chen, Noemi Kedei, Michelle A. Herrmann, Peter M. Blumberg. Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5315. doi:10.1158/1538-7445.AM2014-5315