BackgroundLung adenocarcinoma is the leading cause of cancer-related deaths among both men and women in the world. Despite recent advances in diagnosis and treatment, the mortality rates with an overall 5-year survival of only 15%. This high mortality is probably attributable to early metastasis. Although several well-known markers correlated with poor/metastasis prognosis in lung adenocarcinoma patients by immunohistochemistry was reported, the molecular mechanisms of lung adenocarcinoma development are still not clear. To explore novel molecular markers and their signaling pathways will be crucial for aiding in treatment of lung adenocarcinoma patients.Methodology/Principal FindingsTo identify novel lung adenocarcinoma-associated /metastasis genes and to clarify the underlying molecular mechanisms of these targets in lung cancer progression, we created a bioinformatics scheme consisting of integrating three gene expression profile datasets, including pairwise lung adenocarcinoma, secondary metastatic tumors vs. benign tumors, and a series of invasive cell lines. Among the novel targets identified, FLJ10540 was overexpressed in lung cancer tissues and is associated with cell migration and invasion. Furthermore, we employed two co-expression strategies to identify in which pathway FLJ10540 was involved. Lung adenocarcinoma array profiles and tissue microarray IHC staining data showed that FLJ10540 and VEGF-A, as well as FLJ10540 and phospho-AKT exhibit positive correlations, respectively. Stimulation of lung cancer cells with VEGF-A results in an increase in FLJ10540 protein expression and enhances complex formation with PI3K. Treatment with VEGFR2 and PI3K inhibitors affects cell migration and invasion by activating the PI3K/AKT pathway. Moreover, knockdown of FLJ10540 destabilizes formation of the P110-α/P85-α-(PI3K) complex, further supporting the participation of FLJ10540 in the VEGF-A/PI3K/AKT pathway.Conclusions/SignificanceThis finding set the stage for further testing of FLJ10540 as a new therapeutic target for treating lung cancer and may contribute to the development of new therapeutic strategies that are able to block the PI3K/AKT pathway in lung cancer cells.

Epstein-Barr virus (EBV) expresses an immediate-earlyprotein, Rta, to activate the transcription of EBV lytic genes and the lytic cycle. This work identifies Ubc9 and PIAS1 as binding partners of Rta in a yeast two-hybrid screen. These bindings are verified by glutathione S-transferase pull-down assay, coimmunoprecipitation, and confocal microscopy. The interactions appear to cause Rta sumoylation, because not only can Rta be sumoylated in vitro but also sumoylated Rta can be detected in P3HR1 cells following lytic induction and in 293T cells after transfecting plasmids that express Rta and SUMO-1. Moreover, PIAS1 stimulates conjugation of SUMO-1 to Rta, thus acting as an E3 ligase. Furthermore, transfecting plasmids that express Ubc9, PIAS1, and SUMO-1 increases the capacity of Rta to transactivate the promoter that includes an Rta response element, indicating that the modification by SUMO-1 increases the transactivation activity of Rta. This study reveals that Rta is sumoylated at the Lys-19, Lys-213, and Lys-517 residues and that SUMO-1 conjugation at the Lys-19 residue is crucial for enhancing the transactivation activity of Rta. These results indicate that sumoylation of Rta may be important in EBV lytic activation.Small ubiquitin-like modifiers (SUMOs) 1 are a group of proteins that conjugate a wide range of proteins in the cell (1-3). In human cells, three types of SUMO, i.e. SUMO-1, SUMO-2, and SUMO-3, have been identified (4 -7). These SUMO molecules conjugate to their target proteins through an isopeptide bond formed between the C-terminal glycine residue of SUMO and a lysine residue in the substrate, frequently found at a conserved KXE motif; where represents a hydrophobic amino acid residue, including Leu, Ile, Val, or Phe (8,9). As is generally known, in a SUMO conjugation reaction, SUMO hydrolase first removes the four C-terminal amino acids of SUMO, exposing a glycine residue to facilitate SUMO conjugation. The SUMO molecule is then adenylated and covalently linked to a SUMO-activating E1 enzyme (10, 11). Subsequently, SUMO is transferred to the SUMO-conjugating E2 enzyme, Ubc9, which catalyzes the transfer of SUMO to its target proteins (12-15). The E3 ligase, which stimulates SUMO-1 conjugation to target proteins, has only recently been identified. Three proteins, including PIAS, RanBP2, and Pc2, are currently known to participate in the process of sumoylation (16 -20). Sumoylation may influence protein functions in many ways. An important function of SUMO is to stabilize its target proteins by acting as an antagonist to ubiquitin-mediated proteolysis (21). For instance, SUMO modification blocks ubiquitination and destruction of IB by the SCF(␤-TrCP) E3 ubiquitin ligase complex (21), thus stabilizing the ability of IB to inhibit NF-B. SUMO modification is also known to influence protein localization. For example, SUMO-1 modification not only targets promyelocytic leukemia protein (PML) to discrete subnuclear structures called PML nuclear bodies (22) but also is necessary for RanGAP1 binding t...

Thioridazine (THD) is a common phenothiazine antipsychotic drug reported to suppress growth in several types of cancer cells. We previously showed that THD acts as an antiglioblastoma and anticancer stem-like cell agent. However, the signaling pathway underlying autophagy and apoptosis induction remains unclear. THD treatment significantly induced autophagy with upregulated AMPK activity and engendered cell death with increased sub-G1 in glioblastoma multiform (GBM) cell lines. Notably, through whole gene expression screening with THD treatment, frizzled (Fzd) proteins, a family of G-protein-coupled receptors, were found, suggesting the participation of Wnt/β-catenin signaling. After THD treatment, Fzd-1 and GSK3β-S9 phosphorylation (inactivated form) was reduced to promote β-catenin degradation, which attenuated P62 inhibition. The autophagy marker LC3-II markedly increased when P62 was released from β-catenin inhibition. Additionally, the P62-dependent caspase-8 activation that induced P53-independent apoptosis was confirmed by inhibiting T-cell factor/β-catenin and autophagy flux. Moreover, treatment with THD combined with temozolomide (TMZ) engendered increased LC3-II expression and caspase-3 activity, indicating promising drug synergism. In conclusion, THD induces autophagy in GBM cells by not only upregulating AMPK activity, but also enhancing P62-mediated autophagy and apoptosis through Wnt/β-catenin signaling. Therefore, THD is a potential alternative therapeutic agent for drug repositioning in GBM.

Although prominent FRAT/GBP exhibits a limited degree of homology to Axin, the binding sites on GSK3 for FRAT/GBP and Axin may overlap to prevent the effect of FRAT/GBP in stabilizing beta-catenin in the Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein, GSK3beta interaction protein (GSKIP), which binds to GSK3beta. We have defined a 25-amino acid region in the C-terminus of GSKIP that is highly similar to the GSK3beta interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that GSKIP is a good GSK3beta substrate, and both the full-length protein and a C-terminal fragment of GSKIP can block phosphorylation of primed and nonprimed substrates in different fashions. Similar to Axin GID(381-405) and FRATtide, synthesized GSKIPtide is also shown to compete with and/or block the phosphorylation of Axin and beta-catenin by GSK3beta. Furthermore, our data indicate that overexpression of GSKIP induces beta-catenin accumulation in the cytoplasm and nucleus as visualized by immunofluorescence. A functional assay also demonstrates that GSKIP-transfected cells have a significant effect on the transactivity of Tcf-4. Collectively, we define GSKIP as a naturally occurring protein that is homologous with the GSK3beta interaction domain of Axin and is able to negatively regulate GSK3beta of the Wnt signaling pathway.

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The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. The major need now is to explore this disparate data to find biologically relevant interactions and pathways. Thus, in the post-genomic era, there is clearly a need for the development of algorithms that can accurately predict novel protein-protein interaction networks in silico. The evolutionarily conserved Aurora family kinases have been chosen as a model for the development of a method to identify novel biological networks by a comparison of human and various model organisms. Our search methodology was designed to predict and prioritize molecular targets for Aurora family kinases, so that only the most promising are subjected to empirical testing. Four potential Aurora substrates and/or interacting proteins, TACC3, survivin, Hec1, and hsNuf2, were identified and empirically validated. Together, these results justify the timely implementation of in silico biology in routine wet-lab studies and have also allowed the application of a new approach to the elucidation of protein function in the postgenomic era. Molecular & Cellular Proteomics 3:93-104, 2004.One possible path toward understanding the biological function of a target gene is through the discovery of how it interfaces with known protein-protein interaction networks. We are only now beginning to appreciate the nature and complexity of these networks, and construction of such a network using the traditional biochemical approaches still remains a significant challenge. Recently, the application of high-throughput technologies, such as large-scale yeast twohybrid analysis, has generated an enormous amount of data (1-4). This has led researchers to often face the dilemma of how to effectively utilize the vast information gathered through these large-scale studies. Investigators relying solely on a traditional wet-lab approach for making decisions or setting research priorities are likely to find themselves outpaced by peers who combine in silico biology with empirical methods.

Low response rate and recurrence are common issues in lung cancer; thus, identifying a potential compound for these patients is essential. Utilizing an in silico screening method, we identified withaferin A (WA), a cell-permeable steroidal lactone initially extracted from Withania somnifera, as a potential anti–lung cancer and anti–lung cancer stem-like cell (CSC) agent. First, we demonstrated that WA exhibited potent cytotoxicity in several lung cancer cells, as evidenced by low IC50 values. WA concurrently induced autophagy and apoptosis and the activation of reactive oxygen species (ROS), which plays an upstream role in mediating WA-elicited effects. The increase in p62 indicated that WA may modulate the autophagy flux followed by apoptosis. In vivo research also demonstrated the anti-tumor effect of WA treatment. We subsequently demonstrated that WA could inhibit the growth of lung CSCs, decrease side population cells, and inhibit lung cancer spheroid-forming capacity, at least through downregulation of mTOR/STAT3 signaling. Furthermore, the combination of WA and chemotherapeutic drugs, including cisplatin and pemetrexed, exerted synergistic effects on the inhibition of epidermal growth factor receptor (EGFR) wild-type lung cancer cell viability. In addition, WA can further enhance the cytotoxic effect of cisplatin in lung CSCs. Therefore, WA alone or in combination with standard chemotherapy is a potential treatment option for EGFR wild-type lung cancer and may decrease the occurrence of cisplatin resistance by inhibiting lung CSCs.

Emerging evidence shows that glycogen synthase kinase 3␤ (GSK3␤) is involved in mitotic division and that inhibiting of GSK3␤ kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3␤ involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3␤ interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3␤ and is phosphorylated at Thr-111, Thr-937 ((S/T)P motif) and Ser-974/Thr-978 ((S/T)XXX(S/T)-p motif; p is a phosphorylatable residue). Inhibition of GSK3␤ impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3␤. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3␤. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3␤-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3␤ differentially increases GSK3␤-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3␤ interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3␤-Astrin complex may also facilitate further physiological and pathological phosphorylation.Glycogen synthase kinase 3 (GSK3), 2 a serine/threonine kinase active in several signaling pathways, is involved in the regulation of cell fate, including Wnt and Hedgehog signal transduction, protein synthesis, glycogen metabolism, mitosis and apoptosis (1-4). GSK3 has two structurally similar isoforms in mammals, GSK3␣ and GSK3␤, that are ubiquitously expressed and differ in their N-and C-terminal regions (2, 5). Earlier reports indicated that not only are the developmental profiles of GSK3␣ and GSK3␤ expression different but also the regulation and functions of these two proteins are not always identical (6 -9). Factors known to influence the functions of GSK3␤ include the phosphorylation of GSK3␤ itself, the subcellular localization of GSK3␤, the protein-protein interaction of GSK3␤, and the phosphorylation state of GSK3␤ substrates (1, 3, 4, 10). Insulin-mediated inhibition of GSK3 was mediated through a phosphorylation-dependent mechanism, with the phosphorylation at position Ser-21 and Ser-9 in GSK3␣ and GSK3␤, respectively (11).GSK3␤ also has a preference for pre-phosphorylated substrates, recognizing the consensus sequence (S/T)XXX(S/T)-p. In this sequence the first S/T residue is the target for GSK3␤ phosphorylation, X is any amino acid, P denotes the phosphorylatable residue, and the S/T located at the C terminus is the phosphorylation priming site (12). On the other hand, a number of proteins, including Axin and Tau, are phosphorylated by GSK3␤ without pre-phosphorylation. Recombinant Tau...