Research ArticleFilamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration
Introduction
Caveolin-1 is essential for the formation of caveolae, non-clathrin-coated invaginations and juxtamembrane vesicles of the plasma membrane [1], [2]. As such, it is required for caveolae-mediated functions, notably endocytosis, potocytosis and cholesterol exchange [3], [4], [5], [6]. In addition, caveolin-1 can interact with and modulate the activity of many signaling proteins via a sequence motif called the ‘scaffolding domain’, implicating it as a regulator of signal transduction [7], [8], [9]. In fact, the cellular localization of caveolin-1 is not limited to caveolae, as in various cells it has also been localized to other cellular organelles and compartments [10], [11]. These properties clearly indicate that caveolin-1 is a multifunctional adaptor/scaffold protein with multiple, general as well as cell type-specific, functions.
Early work has implicated caveolin-1 as a tumor suppressor-like protein. This was based on studies showing that caveolin-1 is down-regulated upon oncogenic transformation [12], [13], inhibits mitogenic signaling [7], [13], [14], [15] and abrogates hallmark characteristics of cancer cells such as anchorage-independent growth and invasiveness [16], [17], [18]. Supporting its tumor-suppressor role, caveolin-1 gene knock-out studies have shown that homozygous loss of both caveolin-1 alleles results in sensitization of mammary gland and skin cells to oncogene- and carcinogen-induced tumorigenesis, respectively [19], [20]. However, recent data have pointed to an additional, tumor-promoting action of caveolin-1 in some tumor types and in advanced-stage, metastatic and/or multidrug resistant cancer. Caveolin-1 is highly expressed in many cancer cell lines, as demonstrated initially in human multidrug resistant cancer cells [21], [22] and in mouse metastatic prostate cancer cells [23]. A large body of data that has accumulated in recent years reveals that caveolin-1 is up-regulated in various tumors. Furthermore, the expression of caveolin-1 is positively correlated with the tumors cell grade and their progression stage, and the expression of caveolin-1 may be an independent predictor of poor disease prognosis (reviewed in [24], [25a], [25b]). A possible explanation for the up-regulation of caveolin-1 is that it promotes cancer cell survival. In metastatic prostate cancer cells, caveolin-1 expression and secretion inhibits c-Myc-induced apoptosis and provides an autocrine/paracrine survival signal to androgen-deprived cells [26], [27], [28]. In breast cancer cells, caveolin-1 expression protects the cells from anoikis [18] and abrogates detachment-induced activation of p53 through up-regulation of insulin-like growth factor-I receptors (IGF-IR) and signaling [29], [30]. The inhibitory action of caveolin-1 on anoikis was confirmed in additional cell types [31], [32]. Furthermore, the pro-survival action of caveolin-1 was demonstrated in vivo in a study showing that caveolin-1 gene knock-out stimulates apoptosis in tumor cells derived from the TRAMP mouse prostate cancer model [33].
In addition to its divergent actions on proliferation and survival during oncogenesis and cancer progression, caveolin-1 expression may also affect cancer cell motility during metastasis. Caveolin-1 was recently implicated in the regulation of cell migration [34]. This was mainly studied in endothelial cells, where caveolin-1 is polarized during migration [35], [36], [37], [38] and caveolin-1 knock-down results in loss of directional movement [37], [39]. A stimulatory effect of caveolin-1 on cell migration was also evident in HEK 293T cells stimulated with EGF [40] and in multiple myeloma cells stimulated with VEGF [41]. Although caveolin-1 is known to interact with various proteins that coordinate the re-organization of the actin cytoskeleton (notably, Rho family GTPases [42], [43]), its exact role and mechanism of action in regulating cell migration remained obscure.
In the present study we identify filamin A as a prominent phosphoprotein, previously observed in caveolin-1-expressing stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells [29]. Furthermore, we demonstrate that caveolin-1 expression up-regulates filamin A mRNA and protein levels and that Filamin A is a caveolin-1-dependent target of Akt in IGF-I-stimulated cells. Filamin A is an actin filament cross-linking protein that has been implicated in dynamic remodeling of the actin cytoskeleton network during cell migration, e.g., in cortical neurons and melanoma cells [44], [45]. Our results show that caveolin-1 expression greatly enhances IGF-I-dependent migration of MCF-7 cells by a mechanism that is likely to be mediated by the elevation of filamin A levels and its IGF-I-dependent phosphorylation.
Section snippets
Materials
Unless otherwise stated, all reagents were from Sigma (St. Louis, MO). Protein assays were performed using a modified Lowry procedure with a commercially available kit (Bio-Rad, Hercules, CA). Monoclonal antibodies to caveolin-1 were from Transduction Laboratories (Lexington, KY). Polyclonal antibodies to caveolin-1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to (phospho-Ser-473)-Akt, (phospho-Thr308)-Akt, (phospho-Ser/Thr)-Akt-substrate motif [46a], and
Results
Recently we reported that MCF-7/Cav1 cells (stably transfected MCF-7 human breast cancer cells expressing caveolin-1) exhibit a constitutively phosphorylated Akt kinase and a phosphorylated, high molecular weight, putative Akt substrate protein that we designated pp340, based on its apparent size. In contrast, the parental MCF-7 cells showed little or no pp340 in Western blots, suggesting that the basal phosphorylation state of pp340 is greatly enhanced by the presence of caveolin-1 [29]. We
Discussion
While caveolin-1 is primarily a structural building block of caveolae, it is apparent that this protein also has other cellular roles, consistent with its diverse cellular locations and its multifarious interactions with different proteins [3], [5], [6]. In accord with the multiplicity of functions of caveolin-1 in normal cells, the implications of changes in caveolin-1 expression (or function) for cancer progression are complex and are just beginning to be unraveled [24], [25a]. Early work has
Acknowledgments
We gratefully acknowledge the excellent technical assistance of Yona Ely and we thank all members of our group for their help and support. We are grateful to Prof. Markus Keiser (University of Hannover), Prof. Richard Pagano (Mayo Clinic) and Prof. Christopher McCulloch (University of Toronto) for providing vectors. We thank Prof. Arie Admon (Technion) for mass spectrometry analyses and Dr. Maya Freund (Israel National Center of Forensic Medicine) for cell lines genotyping and authentication.
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