cells, migrate through the interstitial matrix, invade blood and lymph vessels, and deposit into the lymph nodes. Migration and invasion of cells appear to be a result of a complex interplay between the numerous protein families that participate in this process. Mechanisms of cell movement are important not only as part of basic cellular and developmental processes, but also in the pathogenesis of various diseases [5]. To become metastatic, tumor cells must increase the expression of various metastasis-promoting genes. However, in lung cancer, the molecules and mechanisms involved in cell migration or invasion remain largely unknown. Production and secretion of VEGF-A is commonly observed in most aggressive tumors, and expression of VEGF-A profoundly influences the prognosis of cancer patients, including those with lung cancer [6–8]. VEGF-A is one of the most potent stimulators of angiogenesis identified thus far, affecting endothelial cell vascular permeability, proliferation, and motility [7]. Although various intracellular signaling pathways have been proposed to mediate the biological activities of VEGF-A in endothelial cells, the signaling events involved in cell migration and invasion in response to VEGF-A stimulation in lung cancer are not fully understood. FLJ10540 has several names, including CEP55 [9], C10orf3 [10], and URCC6. CEP55 tagged with GFP-C localizes to the centrosome in interphase cells, to the spindle midzone during anaphase, and to the midbody during cytokinesis [9,11–12]. Furthermore, Cdk1, ERK2, and Plk1 cooperate in the phosphor- ylation of CEP55 during mitosis, which is required for the correct mitotic localization of CEP55 and its function during cytokinesis [9]. FLJ10540 is overexpressed during human colon [10] and hepatocellular carcinoma [13] tumorigenesis, suggesting that it may function as an oncogene in tumor development. In addition, we previously showed that the overexpression of FLJ10540 contributes to cellular transformation through the activation of PI3K/AKT [13]. However, no large-scale analysis of FLJ10540 expression and its clinicopathologic and functional significance in human lung cancer has been performed. The aggressive behavior of malignant cancer cells is determined by a complex array of signaling pathways that regulate key functions, such as growth, survival, migration, and invasion. The PI3K/AKT signaling pathway has been causally linked to all four of these responses. [14–17]. Further evidence of the importance of PI3K/AKT signaling in cancer comes from studies which have detected overexpression and hyperactivation of PI3K/AKT in a wide range of human tumors, including lung cancer, and this is often linked with poor prognosis [18]. Accumulating evidence from previous reports suggests a potential role of PI3K/AKT in migration and invasion of various cell types, including lung cancer [19], liver cancer [20], breast cancer [21], and pancreatic cancer [22]. In this study, we show that FLJ10540 is overexpressed in lung adenocarcinoma and that ectopic expression of FLJ10540 promotes cell migration and invasion. In human lung adenocar- cinoma samples, FLJ10540 positively correlated with VEGF-A expression, as determined by gene expression profiling and IHC staining of tissue microarrays. In addition, stimulation with VEGF- A resulted in an increase in FLJ10540 protein expression in a dose- dependent manner in CL1-0 lung cancer cells. Furthermore, FLJ10540 partially translocates from the cytoplasm to the plasma membrane and enhances complex formation with PI3K under VEGF-A stimulation. Finally, VEGF-A affects cell migration and invasion by activating the FLJ10540/PI3K/AKT pathway. These findings suggest that FLJ10540 overexpression is associated with an enhanced metastatic potential and may serve as a potential new therapeutic target for lung adenocarcinoma. VEGFA Upregulates FLJ10540 Results Elevated FLJ10540 expression in lung adenocarcinomas and invasive lung cancer cell lines A significant challenge in the post-genomic era is determining how to prioritize differentially expressed and poorly characterized targets in cancer-related microarray profiling studies. Here, we set up a bioinformatics platform by integrating three different microarray datasets to reveal key regulators involved in the metastasis of lung cancer. Figure 1 shows the schematic data integration approaches and FLJ10540 expression data in different categories. First, samples from 26 patients with lung adenocarci- noma, confirmed by histopathology, were subjected to Affymetrix microarray profiling. Based on the 26 primary lung adenocarci- noma specimens, 826 out of 22,283 differentially expressed transcripts between adjacent non-tumor and tumor areas were clustered by the Wilcoxon signed rank test (p,0.01), based on two- fold differences and tested using the Benjamini and Hochberg false discovery rate correlation (p,0.01). These molecular portraits, containing 192 up-regulated and 634 down-regulated transcripts, could successfully distinguish the patients’ adjacent non-tumor and tumor areas by hierarchical clustering (Figure S1A). Second, public accessible microarray data (downloaded from GEO) were used to acquire different microarray datasets, which were normalized by Quantile Normalization using R to set up potential a metastatic biomarker identification platform. We compared 225 secondary metastatic tumors (including 20 tumor metastases to the lymph node and 200 metastatic tumors) and 30 benign tumors to obtain sets of potential metastatic oncogenes (871) and metastatic suppressor genes (1042). Finally, to prioritize the targets and to validate our metastasis-related genes in cell lines, a series of lung adenocarcinoma cell lines, including CL1-0, CL1-1, CL1-5 and CL1-5- F4, with increasing degrees of invasiveness [23], was also subjected to microarray profiling. A total of 6,238 transcripts had higher expression levels in the high invasion ability cell line CL1-5-F4 than the parental CL1-0 cells. The intersection of these three datasets reveals many novel targets. We characterized the genes that are conserved only in vertebrates, but not in invertebrates, to reveal some novel human cancer-specific abnormalities. As a result, we focused on the poorly characterized gene FLJ10540. FLJ10540 exhibits up-regulated expression patterns in lung adenocarcinomas (Figure 1A, left panel) and metastatic signatures (Figure 1B). To validate the gene expression profiling data for FLJ10540, Q-RT-PCR was performed on 16-pairwise lung adenocarcinoma tumor and adjacent non-tumor samples. Figure S1B shows that overexpression of FLJ10540 was observable in our lung patient samples, with 15 out of 16 lung patient tumors (94%) showing a higher signal after normalization to GAPDH for equal template loading. The average expression level of FLJ10540 in lung adenocarcinomas was 8 fold higher than that in adjacent non-tumor lung tissue samples, as analyzed by boxplot (Figure 1A, right panel). We next verified the expression of FLJ10540 in representative and freshly frozen lung adenocarcinoma and adjacent non-tumor samples by immunohistochemistry staining using anti-human FLJ10540 antibodies. Our data indicate that the level of FLJ10540 was increased in the tumor samples, as compared to that in the adjacent non-tumor tissues (data not shown). Expression of FLJ10540 in a series of invasive human lung adenocarcinoma cancer cell lines During lung tumor development, one of the most critical transitions is the progression from in situ to invasive carcinoma. To explore the possible role of FLJ10540 in the invasiveness of lung adenocarcinoma cells, we first examined the expression of FLJ10540 PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5052