Phosphatidylinositol 3-Kinase dependent upregulation of the epidermal growth factor receptor upon Flotillin-1 depletion in breast cancer cells.

BACKGROUND: Flotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. Recent studies have reported that flotillins promote breast cancer progression, thus making them interesting targets for breast cancer treatment. In the present study, we have investigated the underlying molecular mechanisms of flotillins in breast cancer. METHODS: Human adenocarcinoma MCF7 breast cancer cells were stably depleted of flotillins by means of lentivirus mediated short hairpin RNAs. Western blotting, immunofluorescence and quantitative real-time PCR were used to analyze the expression of proteins of the epidermal growth factor receptor (EGFR) family. Western blotting was used to investigate the effect of EGFR stimulation or inhibition as well as phosphatidylinositol 3-kinase (PI3K) inhibition on mitogen activated protein kinase (MAPK) signaling. Rescue experiments were performed by stable transfection of RNA intereference resistant flotillin proteins. RESULTS: We here show that stable knockdown of flotillin-1 in MCF7 cells resulted in upregulation of EGFR mRNA and protein expression and hyperactivation of MAPK signaling, whereas ErbB2 and ErbB3 expression were not affected. Treatment of the flotillin knockdown cells with an EGFR inhibitor reduced the MAPK signaling, demonstrating that the increased EGFR expression and activity is the cause of the increased signaling. Stable ectopic expression of flotillins in the knockdown cells reduced the increased EGFR expression, demonstrating a direct causal relationship between flotillin-1 expression and EGFR amount. Furthermore, the upregulation of EGFR was dependent on the PI3K signaling pathway which is constitutively active in MCF7 cells, and PI3K inhibition resulted in reduced EGFR expression. CONCLUSIONS: This study demonstrates that flotillins may not be suitable as cancer therapy targets in cells that carry certain other oncogenic mutations such as PI3K activating mutations, as unexpected effects are prone to emerge upon flotillin knockdown which may even facilitate cancer cell growth and proliferation.

A Unidirectional 96-Well Fluidic Culture Platform for Upstream Cell Dosing with Subsequent Downstream Nonlinear and Ascending Exposure Gradients for Real-Time and Cell-Based Toxicity Screening Environments.

Introduction: Because of the importance to create in vitro screening tools that better mimic in vivo models, for exposure responses to drugs or toxicants, reproducible and adaptable culture platforms must evolve as approaches to replicate functions that are native to human organ systems. The Stairstep Waterfall (SsWaterfall) Fluidic Culture System is a unidirectional, multiwell, gravity-driven, cell culture system with micro-channels connecting 12 wells in each row (8-row replicates). Materials and Methods: The construct allows for the one-way flow of medium, parent and metabolite compounds, and the cellular signaling between connected culture wells while simultaneously operating as a cascading flow and discretized nonlinear dosing device. Initial cell seeding in SsWaterfall mimics traditional static plate protocols but thereafter functions with controlled flow and ramping concentration versus time exposure environments. Results: To investigate the utility of a microfluidic system for predicting drug efficacy and toxicity, we first delineate device design, fabrication, and characterization of a disposable dosing and gradient-exposure platform. We start with detailed characterizations by demarcating various features of the device, including low nonspecific binding, wettability, biocompatibility with multiple cell types, intra-well and inter-well flow, and efficient auto-mixing properties of dose compounds added into the platform. Discussion: We demonstrate the device utility using an example in sequential testing-screening drug toxicity and efficacy across wide-ranging inducible exposures, 0 → IC100, featuring real-time assessments. Conclusion: The integrated auto-gradient technology, gravity flow with stairstep pathways, offers end-users an easy and quick alternative to evaluate broad-ranging toxicity of new compound entities (e.g., pharmaceutical, environmental, agricultural, cosmetic) as opposed to traditional/arduous manual drug dilutions and/or expensive robotic technology.

Flotillins bind to the dileucine sorting motif of ß-site amyloid precursor protein-cleaving enzyme 1 and influence its endosomal sorting.

The ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a protease that participates in the amyloidogenic cleavage of the Alzheimer amyloid precursor protein. Trafficking of BACE1 has been shown to be largely mediated by an acidic cluster dileucine motif in its cytoplasmic tail. This sorting signal functions both in endocytosis and endosomal sorting/recycling of BACE1 by providing a binding site for various sorting factors, such as the Golgi-localizing γ-ear containing ADP ribosylation factor binding (GGA) proteins that mediate BACE1 sorting within endosomes. Because flotillin-1 has been suggested to bind to BACE1 cytoplasmic tail, we analyzed the role of flotillins in BACE1 sorting. We show that flotillin-1 directly binds to the dileucine motif in the cytoplasmic tail of BACE1, whereas flotillin-2 binding is mainly mediated by its interaction with flotillin-1. Depletion of flotillins results in altered subcellular localization of BACE1 in endosomes and stabilization of BACE1 protein. Furthermore, amyloidogenic processing of Alzheimer amyloid precursor protein is increased. Flotillins compete with GGA proteins for binding to the dileucine motif in the BACE1 tail, suggesting that they play an important role in endosomal sorting of BACE1. The present study shows for the first time that flotillins are involved in endosomal sorting of BACE1. Because the endosomal localization of BACE1 affects its function as the ß-secretase by increasing amyloidogenic processing of the amyloid precursor protein, flotillins may play a novel role in Alzheimer's disease. The present study is the first to show that flotillins bind to a canonical sorting signal and influence the binding of endosomal sorting factors onto cargo tails.