Elsevier

Lung Cancer

Volume 69, Issue 2, August 2010, Pages 165-171
Lung Cancer

Lipid raft modulation inhibits NSCLC cell migration through delocalization of the focal adhesion complex

https://doi.org/10.1016/j.lungcan.2009.10.014Get rights and content

Abstract

Lipid raft, a specialized membrane structure enriched with cholesterol and glycosphingolipid, contains molecules that convey environmental stimuli to the intracellular systems. Authors investigated the effects of raft cholesterol depletion on non-small cell lung cancer (NSCLC) cell migration. Incubation of NSCLC cells in media containing lovastatin resulted in inhibition of cell migration by 63.1–83.3%, whereas raft cholesterol depletion with successive treatment using methyl-β cyclodextrin (MβCD) followed by lovastatin further suppressed their migration by 35.0–57.8%. Raft cholesterol depletion partially inhibited EGF-induced phosphorylation of EGFR and FAK, however, no change was observed in other molecules comprising focal adhesion complex. It resulted in disappearance of filopodia, inhibition of EGF-induced pY397 FAK aggregation, and its destabilization. Cholesterol depletion inhibited phosphorylation of Src on Y416 in the detergent-insoluble fraction followed by decreased localization of total and pY397 FAK in the detergent-insoluble fraction. Minimal changes in these molecules were observed in the detergent-soluble fraction and interactions between FAK and other molecules of the focal adhesion complex were not influenced. Immunocytochemical analysis confirmed translocation of Src from the raft into cytoplasm and disappearance of EGF-induced membrane ruffling by raft cholesterol depletion. In cholesterol-depleted cells, EGF-induced phosphorylation of Src, Akt, and p44/42 in the detergent-insoluble fraction were inhibited whereas phosphorylation of GSK-3β was unaffected. We conclude that raft cholesterol depletion inhibited NSCLC migration through inhibition of phosphorylation of raft associated Src and dislocation of molecules comprising focal adhesion complexes from raft rather than by inhibiting their recruitment to Src and interaction.

Introduction

Lipid raft is a specialized structure of the plasma membrane containing elevated levels of cholesterol and glycosphingolipids [1], [2]. A variety of proteins are localized in the lipid raft, including caveolins, glycosylphosphatidylinositol (GPI)-anchored protein, EGF receptors, Src family kinases (SFKs), and growth factor receptor-bound protein 2 (Grb2) [3], [4], [5], [6]. The fatty-acid side chains of the phospholipids in lipid raft are more highly saturated and the presence of cholesterol creates a liquid-ordered domain that is less fluidic than the surrounding plasma membrane [1], [2]. These physical properties of lipid raft enable it to serve as signaling platform that colocalizes the requisite components and facilitate their interaction [1].

Adhesion, migration, and metastasis of cancer cells are complex multistep processes requiring a concordant series of signaling events that are organized in time and space. In addition to localization of signaling molecules involved in cell migration, studies from lymphocytes suggest that lipid raft may play important roles in cancer cell migration. Chemoattractants make moving lymphocytes change their morphology into an asymmetric shape with two poles. The acquisition of a polarized shape is accompanied by asymmetric redistribution of membrane receptors and signaling molecules between the L-raft (leading edge), which protrudes at the front of cell, and the U-raft (rear edge), which retracts. The U-raft contains receptors and signaling molecules that are involved in cell adhesion, such as the ezrin, radixin, and moesin (ERM) proteins, CD44, and intercellular adhesion molecules, whereas the L-raft contains the machinery that senses environment stimuli and induces localized actin polymerization [7].

Src, a membrane-associated non-receptor tyrosine kinase, is activated by several types of extracellular signal and regulates the function of a variety of cellular protein substrates [8]. Src and SFKs are the major kinase family associated with lipid raft and have a high binding affinity for phosphorylated focal adhesion kinase (FAK) on Y397. FAK functions as a scaffold to organize structural and signaling proteins within the focal adhesion complex and generates the FAK-Src complex that activates many components of the focal adhesion complex, resulting in initiation of migration cascades [9].

In this study, we investigated the effects of cholesterol depletion in lipid raft on non-small cell lung cancer (NSCLC) cell migration with respect to Src and focal adhesion complexes. Cholesterol depletion in the lipid raft using methyl β-cyclodextrin (MβCD) followed by lovastatin treatment to inhibit accompanying activation of cholesterol biosynthesis is one of commonly used approach to manipulate of raft lipid constituents and disturb its function [10]. Although inhibition of cholesterol biosynthesis made lovastatin used as a tool to disrupt raft [10], lovastatin's migratory inhibiting activities now considered to be originated from diminution of isoprenoid intermediates such as farnesyl-pyrophosphate (PP) and geranylgeranyl-PP, which leads to an inhibition of isoprenylation of small GTPase such as Ras, Rho, Rab and Rap [11]. Combination of MβCD with lovastatin generates rather distinct biologic effect than simple additive effect on lovastatin treated NSCLC cells. In this study, we also routinely include the changes of those molecules from lovastatin treatment alone and compared with those from successive treatment of MβCD and lovastatin. We studied the changes in Src and other molecules involved in focal adhesion in detergent-soluble and -insoluble fractions, and evaluated changes in their interactions related to cholesterol depletion in the lipid raft and morphological changes.

Section snippets

Cell culture and chemical agents

A549, H1299, H460, H157, H358, H1792, and H596 cells were grown in RPMI-1640 media supplemented with 5% fetal bovine serum (FBS) containing penicillin and streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. The following antibodies were used: anti-FAK (A-17), -pY1173 EGFR, -Gαi-3 (C-10), goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, bovine anti-goat IgG-HRP (Santa Cruz Biotechnology, Inc., CA, USA), -paxillin, -pY118 paxillin (BD Bio-sciences, CA, USA), -pS9 GSK-3β, -GSK-3β

Cholesterol depletion in the lipid raft inhibits migration of NSCLC cells

To test the hypothesis that cholesterol modulation of lipid raft is involved in NSCLC cell migration, we first performed a scratch wound healing assay using NSCLC cells treated with 10 μM lovastatin with or without 1% MβCD pretreatment. Five out of seven NSCLC cell lines (A549, H1299, H157, H358, and H596) that were not treated with lovastatin or MβCD briskly migrated into the denuded area resulting in complete closure of the wound within a week. The migratory capacity of these cells was

Discussion

Lipid raft has significant implications for the transmission of stimuli of the extracellular milieu through intracellular signaling systems. Essentially, lipid raft serve as signaling platforms that colocalize the requisite components, facilitating their interaction and supporting signaling. To efficiently transmit environmental stress, the receptors, coupling factors, effector enzymes, and substrates would be colocalized in a single raft [1]. Previous studies on cell migration focused on the

Conflict of interest statement

All authors have none to declare regarding conflict of interest.

Acknowledgements

This study was supported in part by the Institutional Grant from Yonsei University College of Medicine (6-2007) through the Human Barrier Research Institute, Brain Korea 21 Project for Medical Science, and the Korean Science and Engineering Fund through the Cancer Metastasis Research Center at Yonsei University College of Medicine.

References (31)

  • T. Furuchi et al.

    Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK)

    The Journal of Biological Chemistry

    (1998)
  • K. Roepstorff et al.

    Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding

    The Journal of Biological Chemistry

    (2002)
  • E.J. Smart et al.

    Caveolins, liquid-ordered domains, and signal transduction

    Molecular and Cellular Biology

    (1999)
  • A.M. Shenoy-Scaria et al.

    Cysteine 3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae

    The Journal of Cell Biology

    (1994)
  • A. Viola et al.

    Tether and trap: regulation of membrane-raft dynamics by actin-binding proteins

    Nature Reviews: Immunology

    (2007)
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