Loss of profilin-1 expression enhances breast cancer cell motility by Ena/VASP proteins.

Disruption of the actin cytoskeleton is a feature of malignant cells that correlates with dysregulated expression of various actin-binding proteins (ABPs) (Wang et al., 1996; Clark et al., 2000). The malignant phenotype of tumor cells often can be reversed by experimental restoration of ABP expression, suggesting misregulation of ABPs could contribute directly to malignancy (Tanaka et al., 1995; Nikolopoulos et al., 2000). Profilin-1 (Pfn1), a ubiquitously expressed G-actin binding protein, is significantly downregulated in various types of adenocarcinoma (breast, pancreatic, hepatic) (Janke et al., 2000; Gronborg et al., 2006; Wu et al., 2006). This raises a fundamental question as to whether loss of Pfn1 expression contributes to malignant progression of tumor cells. We found that upon downregulation of Pfn1, normal human mammary epithelial cells (HMEC) exhibit disruption of cell–cell adhesion (a hallmark change that facilitates epithelial cell dissemination and migration during cancer progression) (Zou et al., 2007). Furthermore, silencing Pfn1 expression leads to increased motility and invasiveness of breast cancer cell lines and conversely, overexpression of Pfn1 dramatically suppresses the aggressive phenotype of breast cancer cells (Roy and Jacobson, 2004; Zou et al., 2007). These findings collectively suggest that Pfn1 downregulation may contribute to breast cancer invasion and metastasis. Although Pfn1 was initially identified as a G-actin sequestering protein (Karlsson et al., 1977), it can promote actin polymerization by acting as an efficient ADP-to-ATP exchanger on G-actin and adding ATP-G-actin to the barbed but not pointed ends of actin filaments (Witke, 2004). Consistent with this model, Pfn1 depletion induces a significant reduction in F-actin content in various cell types (Ding et al., 2006; Zou et al., 2007). Gene deletion of both Pfn1 and Pfn2 [another variant of Pfn1 that is mainly expressed in the nervous system in vertebrates (Lambrechts et al., 2000)] caused impaired motility of Dictyostelium amebae, providing direct evidence for Pfn’s involvement in migration of eukaryotic cells (Haugwitz et al., 1994). Further support for Pfn1’s requirement in cell migration came from studies that showed cell motility defects in a chickadee (Pfn1 homolog)-null Drososphila mutant (Verheyen and Cooley, 1994) and in human vascular endothelial cells in which Pfn1 expression was silenced (Ding et al., 2006). Several lines of experimental evidence including slower actin-driven intracellular propulsion of bacterial pathogens (Loisel et al., 1999; Mimuro et al., 2000), impaired membrane protrusion of vascular endothelial cells as a consequence of Pfn1 depletion (Ding et al., 2006), and very recently, induction of lamellipodia by Pfn1 in a growth-factor insensitive mechanism (Syriani et al., 2008) all point to a key role of Pfn1 in driving lamellipodial protrusion during cell migration. Several important classes of ABPs that either promote nucleation and/ or elongation of actin filaments at the leading edge including those belonging to Ena/VASP [Enabled/vasodilator stimulated phosphoprotein (Ferron et al., 2007)], Wiskott–Aldrich syndrome protein [WASP; example: Neuronal or N-WASP (Suetsugu et al., 1998), WAVE or WASP-associated verprolin homology (Miki et al., 1998)] family are physiological ligands that bind Pfn1 via polyproline-rich motifs. It has thus been proposed that these proline-rich proteins, when activated by signals, act as scaffolds to spatially recruit Pfn1-actin complex to the sites of actin assembly during cell migration (Holt and Koffer, 2001). In addition to actin and polyproline ligands, Pfn1 also binds to membrane phosphoinositides (phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]), phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3] (Lu et al., 1996). Although the physiological implications of Pfn1’s binding to membrane lipids have not been explored in detail, based on Pfn1’s ability to inhibit PI(4,5)P2 hydrolysis it has been proposed that Pfn1 might also regulate phosphoinositide metabolism in cells (Goldschmidt-Clermont et al., 1990). In fact, a recent study by our group has demonstrated that overexpression of Pfn1 in breast cancer cells dramatically suppresses growth-factor induced PI(3,4,5)P3 generation (Das et al., 2009), Since membrane phosphoinositides are critical regulators of actin cytoskeleton, Pfn1 could be an important nexus between phosphoinositide-derived signaling and cell motility. Given the plethora of evidence demonstrating importance of Pfn1 in driving actin polymerization at the leading edge and hence playing a critical role in cell migration, it is not clear how reduced Pfn1 expression might contribute to faster motility of breast cancer cells. This was the overall goal of the present study where we have now linked loss of Pfn1 expression and hyper-motile phenotype of breast cancer cells through a Ena/VASP-dependent mechanism.