Figure 5. Histopathologic and immunophenotypic findings in lung adenocarcinomas with immunoreactivity for VEGF-A, FLJ10540, and phosphorylated AKT. (A) The tumor has a papillary growth pattern (Hematoxylin & Eosin, 1006). (B) The tumor cells are immunoreactive with VEGF-A, with a staining intensity score of 2 (1006). (C) The tumor cells are immunoreactive with FLJ10540, with a staining intensity score of 2 (1006). (D) The tumor cells are immunoreactive with phosphorylated AKT, with a staining intensity score of 3 (1006). doi:10.1371/journal.pone.0005052.g005 elevated AKT-Ser473 phosphorylation was observed in CL1-0- FLJ10540 and RK3E-FLJ10540 cells, compared with vehicle control cells, whereas FLJ10540-induced AKT-Ser473 phosphor- ylation was completely abolished by LY294002 (Figure 6B). These results indicate that FLJ10540-induced cell migration and invasion is mediated by the PI3K/AKT pathway. We next investigated whether FLJ10540 might serve as a critical mediator of VEGF-A-induced cell migration and invasion. As Figure S3B shows, the FLJ10540-CL1-0 expressing cells, either with or without VEGF-A stimulation, had higher rates of cell migration and invasion, which were also strongly decreased by incubation with LY294002, but not by SB202190 and PD98059. Next, we took two approaches to provide additional evidence for the participation of AKT in FLJ10540- elicited cell migration and invasion upon VEGF-A treatment. First, we analyzed whether the increasing level of endogenous AKT-Ser473 phosphorylation could be observed in CL1-0- FLJ10540 transfected cells upon VEGF-A stimulation. Our data indicated that elevated AKT phosphorylation was observed in CL1-0-FLJ10540 cells upon VEGF-A stimulation, compared with the vehicle treated cells (Figure 6C). Furthermore, FLJ10540- induced AKT-Ser473 phosphorylation was completely abolished by LY294002 upon VEGF-A stimulation (Figure 6C). Our second goal was to examine if specific FLJ10540 siRNAs could affect endogenous AKT activation in CL1-0 cells, with or without VEGF-A stimulation. The data indicated that without VEGF-A stimulation of FLJ10540-depleted cells, the activation of AKT was greatly reduced compared to that seen in negative control cells (Figure 6D, left panel). Compared with negative control cells, the phosphorylated AKT levels were slightly increased upon VEGF-A stimulation in FLJ10540-depleted cells (Figure 6D, right panel), probably due to low residual FLJ10540 present in cells. Taken together, these results indicated that FLJ10540 is required for proper VEGF-A-dependent signaling and contributes to the PI3K/AKT pathway. VEGFA Upregulates FLJ10540 VEGF-A-elicited FLJ0540 expression enhances the physical association with the PI3K complex It has been observed that upon VEGF-A stimulation, increased levels of FLJ10540 can enhance AKT phosphorylation and that FLJ10540 can form a complex with PI3K [13]. This raises the possibility that increased levels of FLJ10540 could reinforce the association between p110-a and p85-a. To test this hypothesis, VEGF-A was used to treat CL1-0 lung cancer cells, either with or without SU5416. Cell lysates were immunoprecipitated with anti- p110-a antibody, and immuno-precipitates were analyzed by immunoblotting with anti-FLJ10540 and anti-p85-a. Cells with increased FLJ10540 levels, due to VEGF-A stimulation, had a slightly enhanced amount of the FLJ10540/p110-a/p85-a com- plex, as compared with cells without VEGF-A treatment (Figure 6E, upper panel). However, the affinity of the FLJ10540/p110-a/p85-a complex was decreased when cells were treated with SU5416 alone or with SU5416 combined with VEGF-A. To further confirm the association between the FLJ10540-PI3K complexes, we used FLJ10540-mediated siRNAs to address this question. Our data indicated that the degree of physical association between p110-a and p85-a was significantly decreased when endogenous FLJ10540 was depleted in CL1-0 lung cancer cells (Figure 6E, lower panel). These data indicated that increased FLJ10540, due to VEGF-A stimulation, may act as an important scaffold protein that can enhance and stabilize the PI3K complex in mammalian cells. Discussion Identification of key regulators from microarray datasets remains a daunting task. Data integration and various bioinfor- matics approaches may substantially facilitate unmasking of a cryptic microarray dataset to identify targets and to dissect underlying mechanisms. Here, we show that FLJ10540 is the molecule interface between VEGF-A and the PI3K/AKT pathway. PI3K/AKT signal transduction has been shown to be a crucial element leading to survival, proliferation, migration, and invasion of human cells induced by VEGF-A [39]. Several reports have shown that the VEGF-A/VEGFR-mediated PI3K/AKT pathway is associated with signaling intermediates, such as phospholipase C-c, GTPase-activating protein, Grb2-Gab1, and Shp2-Gab1 [29,40,41]. These interactions, including several PI3K-interacting partners, result in tyrosine phosphorylation of some of the potential substrates. However, the functional significance between PI3K-associated proteins and PI3K/AKT activation in VEGF-A signaling in lung adenocarcinomas has not been defined clearly. Here, our results show for the first time that FLJ10540 not only forms and stabilizes the PI3K complex upon VEGF-A stimulation, but also contributes to cell migration and invasion following PI3K/AKT activation in lung cancer cells. Our findings unravel a long unanswered question, finally demonstrat- ing that FLJ10540 is the missing factor that bridges the activation mechanism of PI3K/AKT upon VEGF-A stimulation in cancer cells. Moreover, further studies are warranted to investigate whether FLJ10540, translocated from the cytoplasm to the plasma membrane after stimulation by VEGF-A, could interact with VEGFR or VEGFR-associated proteins directly or indirectly, prompting PI3K/AKT activation or participating in other pathways. Various evidence points to the importance of deregulated PI3K/AKT signaling in lung cancer. Overexpression of both, the regulatory p85-a and the catalytic p110-a subunits of PI3K, was reported in primary human lung carcinomas [42,43]. Genomic amplification of the PIK3CA (p110-a) gene was also documented in PLoS ONE | www.plosone.org 8 April 2009 | Volume 4 | Issue 4 | e5052