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

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.

Predicting response to platinum and non-platinum drugs through bioluminescence resonance energy transfer (BRET) based bio-molecular interactions in platinum resistant epithelial ovarian cancer

“…Our NAT and NEO BRET sensors precisely portrayed different ligands (IGF-1, Insulin or EGF) induced differential activation of ERK1/2 and PIP3-AKT in A2780 and MCF7 cell lines with maximal activation caused by IGF-1. Such increased level of AKT activation was earlier reported in MCF7 cells stimulated with IGF-1 compared to EGF stimulation [26] . Presence of specific kinase inhibitors (wortmannin and U0126), RTK inhibitor (PPP) and inactivating mutations (K14A-PH-AKT) reversed the IGF-1 induced increase in NAT and NEO BRET ratio, indicating the specificity of the sensors in dynamic monitoring of these key signalling events.…”

“…The RLuc-PH-AKT/YFP-mem BRET sensor was reported to accurately monitor ligand induced PIP3 formation and AKT activation in MCF7 cells and used as prognostic tool to measure PI3K activity in human serum [26] . Similarly, Rluc-EKAR-Venus (REV), a BRET based ERK1/2 activity reporter was utilized to dynamically monitor ERK1/2 activation in primary cultures of hippocampal neurons isolated from 17 day embryonic mice in response to growth factors and pharmacological inhibitors [ 26 , 27 ]. Though both systems worked suitably on their own, the choice of donor luciferase has been shifted to nanoluciferase in recent years due to its superior brightness and quantum yield.…”

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation

“…The inositol lipid-dependent PM binding of the luciferase linked to the lipid binding domain provided the basis for the non-specific BRET signal generated at the PM. A similar approach was used previously to measure PM PtdIns(3,4,5) P 3 [45], but in that study the co-expression of the BRET pair was provided by the generation of a double stable clone of MCF-7 cells allowing the measurement of the lipid pool shown by Akt-PH, which is known to bind both PtdIns(3,4,5) P 3 and PtdIns(3,4) P 2 [46, 47]. Intermolecular BRET applications become more reliable if the donor and the acceptor molecules are expressed in constant stoichiometric amounts, which is difficult to achieve with co-transfection.…”

Analysis of the ways and methods of signaling pathways in regulating cell cycle of NIH3T3 at transcriptional level

“…RTK could lead to the activation of its downstream molecules SHC, GRB2, RAS, PI3K in turn [ 44 ], and promote to activate PIP2 into PIP3 [ 45 ], and then PIP3 activate APR2/3-F-actin via activating VAV/TIAM [ 46 ], RAC, BAIAP2 and WAVE2 in turn, and promote synthesis of actin [ 47 ]. In this study, VAV family member VAV1 and F - actin family members ACTA1 and ACTG2 were all up-regulated at 25 h, and further analysis of the physiological activities predicted by expression profiles of signaling pathway-associated genes indicated that the synthesis of actin was promoted.…”