The formation of distant metastases is a complex process involving escape of cancer cells from the primary tumor, dissemination to distant organs, and finally re-colonization and expansion (1). For metastatic dissemination, the cancer cell must acquire the ability to migrate, which is associated with cytoskeletal re-arrangements. Migrating cells extend actinbased filopodia and lamellipodia at the leading edge. To initiate this process, a local formation of F-actin is required, which can be mediated by actin nucleating (e.g. Arp2/3 and formins (2)), F-actin bundling (e.g. fascin (3)), and by F-actin cross-linking proteins (e.g. filamins (4)). The actin-severing proteins ADF/ cofilin and gelsolin depolymerize F-actin and thus increase actin turnover (5). The balance between stimulation of actinpolymerizing proteins and actin-depolymerizing proteins is tightly regulated by distinct signaling pathways (e.g. phospholipase C and phosphoinositide 3-kinase (6 -8)). As activation of these pathways can also result in induction of proliferation, "fine-tuning" of continuous signal inputs determines the cellular response. This fine-tuning is mediated by various small molecules, including calcium, cyclic AMP, phosphatidylinositol phosphates, and inositol phosphates. Among the inositides, membranous phosphatidylinositol 4,5-bisphosphate plays a central role in the control of migration as it regulates the activity of cofilin, gelsolin, and profilin (9, 10) and serves as a substrate for the production of the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ).2 Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate increases Ins(1,4,5)P 3 levels and subsequent calcium release from the endoplasmic reticulum. Calcium plays an important role in cell migration because calcium transients activate gelsolin and indirectly ADF/cofilin (9, 10).Inositol 1,4,5-trisphosphate 3-kinase isoenzymes (ITPKA, ITPKB, and ITPKC) metabolize Ins(1,4,5)P 3 to inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ), and thus regulate Ins(1,4,5)P 3 -induced calcium signals (11). The ITPK isoenzymes are highly conserved in their catalytically active C-terminal domains but show large differences in their N-terminal regulatory domains mediating mainly cellular targeting. The isoenzymes differ in subcellular localization and tissue expression patterns. ITPKB and ITPKC mRNAs are ubiquitously expressed, whereas mRNA of ITPKA was only identified in neurons and testis (12). In neurons, ITPKA was shown to be targeted to F-actin via an N-terminal actin binding domain (amino acids 1-66 (13)) and was suggested to be relevant for long term potentiation and spatial learning (14,15
In the present study, effects of increased IP3K-A [Ins(1,4,5)P(3) 3-kinase-A] expression were analysed. H1299 cells overexpressing IP3K-A formed branching protrusions, and under three-dimensional culture conditions, they exhibited a motile fibroblast-like morphology. They lost the ability to form actin stress fibres and showed increased invasive migration in vitro. Furthermore, expression levels of the mesenchymal marker proteins vimentin and N-cadherin were increased. The enzymatic function of IP3K-A is to phosphorylate the calcium-mobilizing second messenger Ins(1,4,5)P(3) to (Ins(1,3,4,5)P(4). Accordingly, cells overexpressing IP3K-A showed reduced calcium release and altered concentrations of InsPs, with decreasing concentrations of Ins(1,4,5)P(3), InsP(6) and Ins(1,2,3,4,5)P(5), and increasing concentrations of Ins(1,3,4,5)P(4). However, IP3K-A-induced effects on cell morphology do not seem to be dependent on enzyme activity, since a protein devoid of enzyme activity also induced the formation of branching protrusions. Therefore we propose that the morphological changes induced by IP3K-A are mediated by non-enzymatic activities of the protein.
Cell migration is one of the hallmarks of metastatic disease and thus identification of migration promoting proteins is crucial for the understanding of metastasis formation. Here we show that the neuron-specific, F-actin bundling inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) is ectopically expressed in tumor cells and critically involved in migration. Down-regulation of ITPKA expression in transformed cell-lines with ectopic expression of ITPKA significantly decreased migration and the number of linear and branched cell protrusion. Conversely, up-regulation of ITPKA in tumor cell lines with low endogenous ITPKA expression increased migration and formation of cell processes. In vitro, ITPKA alone induced the formation of linear actin filaments, whereas ITPKA mediated formation of branched protrusions seems to result from interaction between ITPKA and the F-actin cross-linking protein filamin C. Based on these actin-modulating and migration-promoting effects of ITPKA we examined its expression in clinical samples of different tumor entities, starting with the analysis of multiple tumor tissue arrays. As in lung adenocarcinoma specimens, the highest ITPKA expression rate was found, this tumor entity was examined in more detail. ITPKA was expressed early in adenocarcinoma progression (pN0) and was largely maintained in invasive and metastatic tumor cell populations (pN1/2, lymph node metastases). Together with our result that high expression of ITPKA increases motility of tumor cells we conclude that the observed expression of ITPKA early in tumor development increases the metastatic potential of lung adenocarcinoma cells. Therefore, we suggest that ITPKA may be a promising therapeutic molecular target for anti metastatic therapy of lung cancer.Under physiological conditions, inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA, GeneAtlas, V133A gcma, BioGPS, http:// biogps.gnf.org/#goto¼genereport&id¼3706) is only expressed in hippocampal, cortical and cerebellar neurons.1 In these cells expression of ITPKA increases during brain development 1 and is highest in mature neurons, where the protein accumulates in dendritic spines.2 Through conversion of inositol-1,4,5-trisphosphate (InsP 3 ) to inositol-1,3,4,5-tetrakisphosphate (InsP 4 ) ITPKA is an important regulator of InsP 3 induced calcium signaling in dendritic spines. 2,3 In addition to this catalytic activity, it has been shown that ITPKA has Factin bundling activity. It bundles actin filaments by formation of homodimers and binding to F-actin, making ITPKA to an important regulator of spine morphology. 4 Beside this physiological role, we recently have found a pathological function of ITPKA in tumor cells. We revealed that particular tumor cell lines express ITPKA and demonstrated that this ectopic expression of ITPKA increased the metastatic potential of tumor cells. Because of its F-actin bundling activity, ITPKA induces the formation of filopodia-and lamellipodia-like protrusions, 5,6 which are critical for cells to migrate. 7 This nonenzymatic migration-prom...
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