VEGFA Upregulates FLJ10540 Figure 3. The migration and invasion abilities of lung cancer cells are inhibited by FLJ10540-mediated siRNAs. (A and B, left panel) A negative control siRNA with two different FLJ10540 siRNAs (siFLJ10540-1 and siFLJ10540-2) were transfected into CL1-5 and H1299 cells for 24 hours. After transfection, western blotting was performed using anti-FLJ10540 and anti-b-actin antibodies. (A and B, middle panel) To measure cell growth rates, 56103 cells of negative control-CL1-5, CL1-5-FLJ10540 siRNAs, negative control-H1299, and H1299-FLJ10540 siRNAs were plated at day 0 in 96 well plates with 10% FBS. The cells were counted at indicated time points by the MTT assay (OD570) to quantify the cell growth. Data were normalized against the OD570 value on day 0 of each treatment. The growth rate of CL1-5 and H1299 cells are shown as the mean6s.d. of three independent experiments. (A and B, right panel) The migration relative-fold and the invasion relative-fold of CL1-5 and H1299 were normalized against negative control cells and represented diagrammatically. The results represent the mean6s.d. of three independent experiments. doi:10.1371/journal.pone.0005052.g003 induced EMT, thereby enhancing cell migration and invasion, we used H1299 and CL1-0 lung cancer cells, with or without VEGF-A stimulation, to address this question. However, despite increasing cell motility by overexpressing FLJ10540 or decreasing cell motility by FLJ10540-mediated siRNAs, differences in FLJ10540 levels did not in any way alter the expression levels of any of the marker proteins associated with EMT, such as E-cadherin and vimentin (data not shown). Therefore, FLJ10540 seems to mediate migration and invasion effects of VEGF-A independently of EMT and does not appear to play a role in the regulation of EMT, at least in the lung cancer cell lines we analyzed. A positive correlation in protein expression of FLJ10540/ VEGF-A and FLJ10540/AKT-p is observed in clinical specimens of lung adenocarcinoma at variable stages VEGF signals are partially tramitted through a PI3K-mediated signaling cascade, which includes AKT [38], and FLJ10540 could form a complex with PI3K in HCC [13]. These observations raise the possibility that VEGF-A/FLJ10540/PI3K/AKT might be in the same pathway, despite the fact that the gene expression patterns of AKT in our lung cancer microarray dataset did not exhibit any correlation with FLJ10540. This is not surprising, because the activation state of the PI3K signaling pathway is monitored by phosphorylated AKT. Therefore, we set up a tissue microarray, containing 273 archived lung adenocarcinomas at variable stages, to examine the protein expression patterns among these molecules. The immunohistochemical studies of HE staining (Figure 5A), VEGF-A (Figure 5B), FLJ10540 (Figure 5C), and phosphorylated AKT (Figure 5D) were performed on these lung adenocarcinoma specimens. The correlation between each paired IHC score of AKT-p, VEGF, and FLJ10540 was analyzed by the Spearman’s rank tests. The results showed that there were positive correlations between VEGF and FLJ10540 (correlation coeffi- cient = 0.36, p,0.001) and AKT-p and FLJ10540 (correlation coefficient = 0.22, p = 0.001). These results seem to imply that FLJ10540 may participate in a VEGF-A-mediated PI3K/AKT pathway in lung adenocarcinoma. Activation of PI3K/AKT is critical for FLJ10540-induced lung cancer cell migration and invasion upon VEGF-A stimulating Tumor cells possess a broad spectrum of migration and invasion mechanisms that are associated with enhanced tumor metastasis [4]. To explore how many signaling transduction pathways might participate in FLJ10540-mediated cell migration and invasion in lung cancer cells, we first investigated three well- defined signaling pathways, namely the PI3K/AKT, p38, and MAPK pathways, using pharmacological inhibitors. Figure 6A and Figure S3A show that FLJ10540-CL1-0 and FLJ10540- RK3E expressing cells had increased levels of cell migration and invasion, which were strongly decreased by incubation with LY294002, and to a much lesser extent, with SB202190 and PD98059. The same was found when PI3K was inhibited with wortmannin, a PI3K inhibitor (data not shown). Moreover, PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5052