Abstract 3835: Multiple stimulants target different phosphoinositide 3-kinase (PI3K) classes in breast cancer cell lines

Abstract

Abstract Phosphoinositides (PIPs) are an integral part of multiple cell signaling pathways and are metabolized by kinases, phosphatases, and phospholipases. PIPs produced by the activities of phosphoinositide 3-kinase (PI3K) serve as second messengers in cell survival, growth, motility, immune response, inflammation, and apoptosis. Conversely, abnormal production of these signaling lipids leads to proliferative, metabolic, and inflammatory disorders. It is critical to know the absolute and relative concentration of individual PIPs in cell samples, however this analysis is difficult due to their low abundance and the inherent physical properties of lipids. Experiments to measure PIPs have involved radiolabeling cells, extraction of radioactive products, and separation using thin-layer chromatography. The amount of PIPn extracted from cell lysates was determined by means of a standard competitive ELISA format, eliminating the need for radioactivity. Using specific antibodies and binding proteins we developed assays able to differentiate closely related PIPs. These nonradioactive assays employ synthetic di-C16 lipids as standards and are able to measure as little as 0.1 pmol of lipid. Cell-extracted PIPs were added as competitors in these quantitative ELISAs. We used the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 (PTEN deficient) as a model system to evaluate multiple activators of PI3K pathways by looking at levels of PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. Using growth factors, hyperosmolarity, and oxidative stress we found that different stimulants targeted different classes of PI3K. All three classes of PI3K were more active in PTEN deficient cell line (468 cells) than PTEN+/+ (231 cells). We found that all stimulants used increased PI3K products. Pre-incubation with wortmannin, a known PI3K inhibitor, decreased PIPn levels. We propose that hyperactivation of class I and class II PI3K in the absence of PTEN regulation (468 cells) leads to excessive 3’ phosphorylated phosphoinositide levels that are known to activate Akt, We expect the ability to quantify individual phosphoinositides from cells will help assign specific biological functions to these important lipid second messengers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2011-3835