Ye et al. BMC Cancer 2010, 10:611 http://www.biomedcentral.com/1471-2407/10/611 Page 9 of 11 Figure 4 Effect of Plexin-B1 inhibition on cytoskeleton rearrangement. Representative cytoskeletal rearrangements (phalloidin-fluorescein isothiocyanate staining) of untransfected (Blank control) SKOV3 cells and SKOV3 cells at 24 h after a 5-h exposure to Plexin-B1 siRNA2, Plexin-B1 siRNA3 or negative control siRNA. Repression of Plexin-B1 by Plexin-B1 siRNA2 and siRNA3 inhibited F-actin polymerization and filopodia formation in SKOV3 cells. Red indicates F-actin. Recent studies have provided evidence that, in response to Sema 4D, Plexin-B1 stimulates endothelial cell migration by activating the PI3K/AKT signaling pathway [3,41]. The present work showed that reducing Plexin-B1 expression in SKOV3 cells inhibited AKT activity by reducing the level of phospho-AKT (Ser473), which plays important roles in regulating cell prolifera- tion, migration and invasion. However, the proliferation rate of SKOV3 cells was not affected by Plexin-B1 knock-down, suggesting that Plexin-B1 has no essential role in regulating the proliferation of SKOV3 cells. Swiercz et al. [26] demonstrated that Plexin-B1 activates the the PI-3-kinase/Akt pathway in response to Sema 4D, but requires ErbB-2 to activate RhoA and conse- quently promote cell migration. Interestingly, they also suggested that Plexin-B1 inhibits RhoA activity in the presence of Met, resulting in an antimigratory effect. They proposed a novel mechanism by which Plexin-B1- mediated signaling can be regulated via differential asso- ciation with ErbB-2 and Met, and the cellular effects elicited by Plexin-B1 are hypothesized to be determined by the relative expression levels of ErbB-2 and Met, which compete to mediate Sema 4D’s effects. In our study, the SKOV3 cells expressed both Met and ErbB-2 [42,43], and inhibition of Plexin-B1 resulted in reduc- tions in cell motility and invasive capacity. These results indicate that Plexin-B1 may principally interact with ErbB-2 to activate AKT in SKOV3 cells and conse- quently stimulate the PI3K/AKT pathway, which plays a key role in the progression and metastasis of various human ovarian cancers [35]. To date, Plexin-B1 has been shown to induce the reor- ganization of the cytoskeleton by stimulating different downstream adaptors. On the one hand, dimerization of Plexin-B1 is sufficient to induce ErbB2-dependent activation of Rho and to regulate the remodeling of the actin cytoskeleton and alter cell movement in response to Sema 4D [22]. On the other hand, it has been shown that Plexin-B1 inactivates PI3K, dephosphorylates AKT and activates GSK-3b through R-Ras GAP activity. Acti- vation of GSK-3b leads to inhibition of microtubule poly- merization and stabilization [44]. In this study, we found that effective suppression of Plexin-B1 in SKOV3 cells led to cell shrinkage and loss of polarity without F-actin polymerization or filopodia formation. The loss of cell polarity in terms of actin assembly and stable microtu- bule distribution suggested that there should also be defects in cell migration and invasion [45]. Furthermore, several other regulators of the actin cytoskeleton may also be implicated in the Plexin-B1-induced reorganiza- tion of SKOV3 cells. For example, the MEK/ERK signal- ing pathway, which was reported to be constitutively activated in epithelial ovarian cancer cells [46], has been shown to restore stress fibers in transformed fibroblasts [47], and a recent study suggested that Plexin-B1 can uti- lize RhoA to stimulate ERK [48]. However, in this report, it remains uncertain whether the reduced AKT activity induced by Plexin-B1 inhibi- tion and the corresponding cellular responses are the result of Sema 4D produced by SKOV3 cells acting on the Plexin-B1 receptor to stimulate downstream mole- cules via an autocrine/paracrine mechanism, or whether overexpression and consequent clustering of Plexin-B1 in SKOV3 cells leads to phosphorylation of its cytoplas- mic region. We have produced a Sema 4D fusion pro- tein in vitro and further investigations are under way. In addition, studies addressing whether this oncogenic function of Plexin-B1 in SKOV3 cells is a cell type-spe- cific effect or instead is shared across multiple ovarian cancer cell lines are ongoing.