Lipid Raft Association Restricts CD44-Ezrin Interaction and Promotion of Breast Cancer Cell Migration

Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration.The membrane protein CD44 is a multifaceted molecule involved in many different cellular processes, including organ development, neuronal axon guidance, immune functions, hematopoiesis, and migration.^1–4 It acts as a receptor for the extracellular matrix component hyaluronic acid (HA)^5,6 and for the secreted extracellular protein osteopontin.⁷ CD44 is an important mediator of cellular adhesion and migration due to its active influence on the organization of the actin cytoskeleton. This occurs through direct interactions between CD44 and different actin-binding partners, of which the most common are proteins belonging to the ezrin-moesin-radixin (ERM) family. The ERM proteins form a bridge between CD44 and the actin cytoskeleton, mediating cell morphology changes that are important for cell migration. Ezrin interacts with CD44 and F-actin, respectively, through its conserved N-terminal band four-point-one, ezrin, radixin, moesin domain and C-terminal ERM Association Domain domain. In the inactive configuration of ezrin, both domains interact with each other and block the binding sites for CD44 and F-actin. Ezrin activation is mediated by phosphorylation-induced conformational changes,⁸ with phosphorylation on threonine-567 being necessary for binding to the F-actin cytoskeleton.⁹Phosphorylation of CD44 has also been shown to be important for its activation, particularly on serine residues in the C-terminal domain.^8,10 CD44 has been described to be enriched in cholesterol- and sphingomyelin-enriched membrane microdomains termed lipid rafts.¹¹ Much evidence has suggested the involvement of lipid rafts in regulating different cellular events, including migration.¹² Because some of these cellular events are frequently altered in cancer, it has been hypothesized that lipid rafts play a crucial role in regulating cancer progression.¹³ However, although alterations in CD44 expression have been associated with many cancers,¹⁴ how lipid rafts influence the subcellular localization (and thus migratory functions) of CD44 and its contribution toward cancer progression is not well understood.Whether or not CD44 and its binding partners localize to lipid rafts may in fact regulate several signaling cascades. CD44 is usually directed toward lipid rafts via posttranslational lipid modifications called acylation reactions, the most common of which is palmitoylation. Due to its dynamic and reversible nature, palmitoylation can have important functions in dictating protein fate such as protein trafficking, lateral segregation, and cellular localization. Palmitoylation plays an important role in CD44-HA turnover, with palmitoylated CD44 promoting CD44-HA endocytosis. Accordingly, lipid rafts have been described to play an important role in cellular endocytosis.¹⁵Ezrin localization to lipid rafts is controversial¹⁶ and the mechanisms regulating its affiliation with lipid rafts incompletely understood. Ezrin interactions with phosphatidylinositol 4,5-biphosphate (PIP2) may be important for its activation, causing the four-point-one and C-terminal ERM domains to open¹⁷ and permitting ezrin localization at the plasma membrane.¹⁸ Because PIP2 has been described to be enriched in lipid rafts,¹⁹ it is possible that ezrin localizes to lipid rafts through an interaction with PIP2.In this paper, we set out to investigate the role of lipid rafts in regulating CD44-dependent breast cancer cell migration. Our initial findings revealed that CD44 and ezrin localized to different membrane fractions in nonmigrating cells, biochemically characterized as lipid raft and nonraft domains, respectively. In response to migratory stimuli (either random or CD44-specific), the proportion of raft-affiliated CD44 decreased whereas that of ezrin did not change. Moreover, under migrating compared to nonmigrating conditions, immunofluorescence confocal microscopy revealed increased colocalization of CD44 with the nonraft marker transferrin receptor. Altogether, we present novel evidence that physical interactions between CD44 and ezrin occur in nonraft fractions of migrating cells. In support of our observations, pharmacological disruption of lipid rafts increased CD44-ezrin coprecipitation, whereas enhanced retention of CD44 within rafts abolished CD44-ezrin coprecipitation. Surprisingly, flotillin-1 or caveolin-1 transient knockdown alone did not affect cell migration in these cells, suggesting compensatory mechanisms that make up for the presumed loss of one or other raft compartment. In support of this assumption, coincident knockdown of flotillin-1 and caveolin-1 significantly impaired cell migration. Nonetheless, our data are consistent with a novel regulatory mechanism in which CD44 translocates outside lipid rafts to bind ERM binding partners such as ezrin and drive cell migration. Future exploration of the precise mechanisms regulating this translocation may reveal future targets for interfering with breast cancer cell migration during the early stages of metastasis.