Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP3) Biosensor

Vittorio De Franciscis,Mei-Shiue Kuo,Tarik Issad,Cécile Pierre-Eugène,Johanna Auriau

doi:10.1371/journal.pone.0092737

Abstract

Stimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP2 to generate PIP3, which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP3 production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP3 production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP3 and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer.

Highlights

The PI3K/Akt pathway regulates multiple biological processes such as metabolism, cell proliferation, survival, migration and apoptosis [1,2]
To verify that expression of Luc-Akt-pleckstrin homology (PH) and YFPMem did not alter the activity of the PI3K/Akt signaling pathway, we compared Akt phosphorylation in Luc-Akt-PH and B2 clones with the parental MCF-7 cell line upon insulin stimulation
These results demonstrate that the MCF-7/B2 clone can be used to evaluate PIP3 stimulatory activity present in human serum, and that in the commercial batch of human serum used in this study, this stimulatory effect is mediated by a heat-sensitive molecule with a molecular weight higher than 3 kDa, the activity of which is inhibited by the presence of IGFBP1, suggesting that IGF1 and/or IGF2 could be the factors responsible for activation of the PI3K pathway

Summary

Introduction

The PI3K (phosphatidylinositol 3-kinase)/Akt pathway regulates multiple biological processes such as metabolism, cell proliferation, survival, migration and apoptosis [1,2]. Tyrosine kinase receptors (RTKs) are activated and autophosphorylate on tyrosine residues that serve as docking sites for a number of Src homology 2 (SH2) domain-containing proteins, such as the p85 regulatory subunit of PI3K. P85 can interact indirectly with RTKs through binding of its SH2 domains to tyrosine phosphorylated residues on adaptor proteins, such as IRSs (Insulin Receptor Substrates). The engagement of p85 to activated receptors induces conformational changes that relieves the intermolecular inhibition of the p110 catalytic subunit and brings it near to its plasma membrane lipid substrate Phosphatidyl Insositol Phosphate 2 (PIP2), which is phosphorylated to produce PIP3 [3]. PIP3 recruits PDKs (3-phosphoinositide-dependent protein kinases) and Akt to the plasma membrane via their PH domains, where they are subsequently phosphorylated and activated [1,2]. PTEN (phosphatase and tensin homologue deleted on chromosome 10) terminates the PI3K/Akt signaling by dephosphorylating PIP3 into PIP2 [4]

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Results

Conclusion