Development of a human breast-cancer derived cell line stably expressing a bioluminescence resonance energy transfer (BRET)-based phosphatidyl inositol-3 phosphate (PIP3) biosensor.

International audienceStimulation 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

Effect of inhibitors of the PI3K/Akt signaling pathway on BRET signal in MCF-7/B2 cells.

<p>(A) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 20 ng/μl of the humanized anti-EGFR antibody Cetuximab. Cells were then stimulated with EGF (32 nM), and light emission acquisition started immediately. A typical real-time BRET experiment (left panel) and the mean ± SEM of BRET values at the peak (right panel) are shown (n = 3). (B) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 25 μM of the tyrphostin AG1024. Cells were then stimulated with 100 nM insulin, and light emission acquisition started immediately. A typical real-time BRET experiment (left panel) and the mean ± SEM of BRET values at the plateau (right panel) are shown (n = 3). (C) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 25 μM of the PI3K inhibitor LY294002. Cells were then stimulated with 10 nM IGF1 and light emission acquisition started immediately. A typical real-time BRET experiments (left panel) and the mean ± SEM of BRET values at the plateau (right panel) of 3 independent experiments are shown. (D, E) MCF-7/B2 cells were preincubated for 4 h in absence or presence of 10 μM of the inhibitors of Akt-PH/PIP₃ interaction PIT-1 (D) and DMPIT-1 (E). Cells were then stimulated with 10 nM IGF1, and light emission acquisition started immediately. Typical real-time BRET experiments (left panels) and the mean ± SEM of BRET values at the plateau (right panels) of 3 to 4 independent experiments are shown. Statistical analysis was performed using ANOVA followed by Tukey’s test. *, P<0.05; **, P<0.01; ***, P<0.001; NS, Non significant.</p

The PI3K stimulatory activity present in human serum can be evaluated using the MCF-7/B2 cells.

<p>(A, B) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with PBS containing 0%, 1%, 2%, 5% or 10% human serum that had been previously heated or not at 95°C during 1 h, and light emission acquisition started immediately. (A) A typical real-time BRET experiment is shown. (B) Means ± SEM of BRET values at the plateau of 3 to 7 independent experiments are shown. (C) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with 5% human serum previously submitted or not to centrifugation on a centrifugal filter device with a molecular weight cut-off of 3 kDa. A typical real-time BRET experiment (left panel) and means ± SEM of BRET values at the plateau (right panel) are shown (n = 3). (D) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with 5% human serum that had been pre-incubated for 1 h in presence of 50 μM IGFBP1. A typical real-time BRET experiment (left panel) and means ± SEM of BRET values at the plateau (right panel) are shown (n = 3). Statistical analysis was performed using ANOVA followed by Tukey’s test. *, P<0.05; **, P<0.01; ***, P<0.001; NS, Non significant.</p

Effect of IGF1, IGF2, EGF and glargine on PIP₃ production in MCF-7/B2 cells.

<p>(A) Typical experiments showing real-time effects of IGF1, IGF2, EGF and glargine on PIP₃ production in MCF-7/B2 cells. (B) Dose-dependent effect of IGF1, IGF2, EGF, glargine and insulin on PIP₃ production in MCF-7/B2 cells. Ligand-induced BRET (BRET above basal) at the plateau (IGF1, IGF2, Insulin, Glargine) or at the peak (EGF) was determined for each ligand concentration and was used to establish dose-response curves. Results are means ± S.E.M. of 3 to 8 independent experiments. EC50 for insulin, IGF1, IGF2, EGF and glargine are given in the result section.</p

Development of a human breast cancer-derived clone stably expressing a BRET-based PIP₃ biosensor.

<p>(A) Principle of the BRET-based assay used to monitor PIP₃ production in living cells. Activation of the PI-3 kinase by tyrosine kinase receptors induces phosphorylation of PIP₂ into PIP₃ and recruitment of Akt to the plasma membrane through its PH domain. To monitor PIP₃ production, MCF-7 cells were stably transfected with the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to a membrane localization sequence (YFP-membrane). Recruitment of Luc-Akt-PH to the plasma membrane by PIP₃ results in energy transfer between Luc-Akt-PH and YFP-membrane. This permits to study the pharmacological properties of ligands that activate this pathway, and to evaluate the effects of inhibitory molecules acting on (I) the extracellular part of receptors, (II) the tyrosine kinase activity of the receptors, (III) the catalytic activity of the PI3K, and (IV) the interaction between PIP₃ and the PH domain of Akt. (B) Transfection of a MCF-7 clone stably expressing Luc-Akt-PH with YFP-membrane cDNA gave rise to 4 sub-clones stably expressing both constructs. Ligand-induced BRET could be detected in only 3 sub-clones (B2, D2, C2). Preliminary BRET experiments indicated that insulin-induced BRET was higher with clone B2. (C) Surface expression of pEYFP-Mem in MCF-7/B2 cells was studied by fluorescence microscopy. YFP fluorescence was detected using a FITC filter and nuclei were visualized using a DAPI filter. The image was obtained by deconvolution analysis. (D) Western-blotting experiment showed that expression of Luc-Akt-PH alone or together with YFP-membrane does not affect insulin-induced phosphorylation of endogenous Akt. (E) Insulin dose-dependently stimulated PIP₃ production in MCF-7/B2 cells. Left panel: typical real-time experiment showing basal and insulin-stimulated BRET. Right panel: insulin-induced BRET (BRET above basal at the plateau) was determined for each insulin concentration to establish dose-response curves. Results are means ± S.E.M. (standard error of the mean) of 6 to 11 independent experiments. The freeze-thaw cycle did not affect the sensitivity of the cells to insulin stimulation.</p

Apelin affects the mouse aging urinary peptidome with minimal effects on kidney

International audienceKidney function is altered by age together with a declined filtration capacity of 5-10% per decade after 35 years. Renal aging shares many characteristics with chronic kidney disease. Plasma levels of the bioactive peptide apelin also decline with age and apelin has been shown to be protective in chronic kidney disease. Therefore we evaluated whether apelin could also improve aging-induced renal lesions and function in mice. Since urine is for the major part composed of proteins and peptides originating from the kidney, we first studied apelin-induced changes, in the aging urinary peptidome. Despite the recently published age-associated plasma decrease of apelin, expression of the peptide and its receptor was increased in the kidneys of 24 months old mice. Twenty-eight days treatment with apelin significantly modified the urinary peptidome of 3 and 24 months old mice towards a signature suggesting more advanced age at 3 months, and a younger age at 24 months. The latter was accompanied by a decreased staining of collagen (Sirius red staining) in 24 months old apelin-treated mice, without changing aging-induced glomerular hypertrophy. In addition, apelin was without effect on aging-induced renal autophagy, apoptosis, inflammation and reduced renal function. In conclusion, treatment of aged mice with apelin had a limited effect on kidney lesions although modifying the urinary peptidome towards a younger signature. This supports evidence of apelin inducing more general beneficial effects on other aging organs, muscles in particular, as recently shown for sarcopenia, markers of which end up via the glomerular filtration in urine

Upregulated Apelin Signaling in Pancreatic Cancer Activates Oncogenic Signaling Pathways to Promote Tumor Development

International audienceDespite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated β-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease

SHP2 drives inflammation-triggered insulin resistance by reshaping tissue macrophage populations

International audienceHyperactive SHP2 promotes inflammation-driven insulin resistance by skewing macrophage balance in metabolic tissues and may be a target in T2D