Expression of DIAPH1 is up-regulated in colorectal cancer and its down-regulation strongly reduces the metastatic capacity of colon carcinoma cells.

In most cases, metastatic colorectal cancer is not curable, thus new approaches are necessary to identify novel targets for colorectal cancer therapy. Actin-binding-proteins (ABPs) directly regulate motility of metastasising tumor cells, and for cortactin an association with colon cancer metastasis has been already shown. However, as its depletion only incompletely inhibits metastasis, additional, more suitable cellular targets have to be identified. Here we analyzed expression of the ABPs, DIAPH1, VASP, N-WASP, and fascin in comparison with cortactin and found that, besides cortactin, DIAPH1 was expressed with the highest frequency (63%) in colorectal cancer. As well as cortactin, DIAPH1 was not detectable in normal colon tissue and expression of both proteins was positively correlated with metastasis of colorectal cancer. To analyse the mechanistic role of DIAPH1 for metastasis of colon carcinoma cells in comparison with cortactin, expression of the proteins was stably down-regulated in the human colon carcinoma cell lines HT-29, HROC-24 and HCT-116. Analysis of metastasis of colon carcinoma cells in SCID mice revealed that depletion of DIAPH1 reduced metastasis 60-fold and depletion of cortactin 16-fold as compared with control cells. Most likely the stronger effect of DIAPH1 depletion on colon cancer metastasis is due to the fact that in vitro knock down of DIAPH1 impaired all steps of metastasis; adhesion, invasion and migration while down-regulation of cortactin only reduced adhesion and invasion. This very strong reducing effect of DIAPH1 depletion on colon carcinoma cell metastasis makes the protein a promising therapeutic target for individualized colorectal cancer therapy.

Tumour cells can employ extracellular Ins(1,2,3,4,5,6)P(6) and multiple inositol-polyphosphate phosphatase 1 (MINPP1) dephosphorylation to improve their proliferation.

InsP(6) [Ins(1,2,3,4,5,6)P6; phytate] is the most abundant inositol phosphate in mammalian cells with cytosolic/nuclear concentrations of up to 50 µM. We noticed that InsP6 in culture medium at a concentration of ≤50 µM significantly stimulates H1299 tumour cell growth, whereas larger concentrations of InsP6 inhibit growth. A detailed study of the fate of 30 µM InsP6 added to H199 cells revealed a major fraction of InsP6 initially precipitates as cell-surface metal complexes, but becomes slowly re-solubilized by extracellular dephosphorylation first to InsP3 isomers and subsequently to free myo-inositol. The precipitated metal-InsP6 complex is endocytosed in a receptor-independent but intact-glycocalyx-dependent manner and appears in lysosomes, where it is immediately dephosphorylated to Ins(1,2,4,5,6)P5 and very slowly to free inositol. By RNA knockdown, we identified secreted and lysosome targeted MINPP1 (multiple inositol-polyphosphate phosphatase 1), the mammalian 3-phytase, to be essentially involved both in extracellular and in lysosomal InsP6 dephosphorylation. The results of the present study indicate that tumour cells employ this enzyme to utilize the micronutrients myo-inositol and metal-phosphate when encountering extracellular InsP6 and thus to enhance their growth potential.

DIAPH1 facilitates paclitaxel-mediated cytotoxicity of ovarian cancer cells.

The chemotherapeutic agent paclitaxel (PTX) selectively binds to and stabilizes microtubule (MTs). Also, the activated formin Diaphanous Related Formin 1 (DIAPH1) binds to MTs and increases its stability. In a recent study, we found that high DIAPH1 levels correlated with increased survival of ovarian cancer (Ovca) patients. A possible explanation for this finding is that Ovca cells with high DIAPH1 levels are more sensitive to PTX. To examine this assumption, in this study the effect of DIAPH1 depletion on PTX-mediated cytotoxicity of OVCAR8 and OAW42 cells was analyzed. Our data showed that down-regulation of DIAPH1 expression decreased PTX sensitivity in both cell lines by reducing apoptosis or necrosis. Analysis of MT stability by Western blotting revealed a decreased concentration of stable, detyrosinated MTs in PTX-treated DIAPH1 knock-down compared to control cells. Also, in fixed metaphase cells the level of stable, detyrosinated spindle MTs decreased in cells with reduced DIAPH1 expression. In vitro analysis with recombinant DIAPH1 protein showed that PTX and DIAPH1 exhibited additive effects on MT-polymerization, showing that also in a cell-free system DIAPH1 increased the effect of PTX on MT-stability. Together, our data strongly indicate that DIAPH1 increases the response of Ovca cells to PTX by enhancing PTX-mediated MT-stability.

Inositol 1,4,5-trisphosphate 3-kinase-A is a new cell motility-promoting protein that increases the metastatic potential of tumor cells by two functional activities.

Cellular migration is an essential prerequisite for metastatic dissemination of cancer cells. This study demonstrates that the neuron/testis-specific F-actin-targeted inositol 1,4,5-trisphosphate 3-kinase-A (ITPKA) is ectopically expressed in different human tumor cell lines and during tumor progression in the metastatic tumor model Balb-neuT. High expression of ITPKA increases invasive migration in vitro and metastasis in a xenograft SCID mouse model. Mechanistic studies show that ITPKA promotes migration of tumor cells by two different mechanisms as follows: growth factor independently high levels of ITPKA induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton. The F-actin binding activity of ITPKA stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin. In growth factor-stimulated cells, the catalytically active domain enhances basal ITPKA-induced migration by activating store-operated calcium entry through production of inositol 1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol phosphate 5-phosphatase. These two functional activities of ITPKA stimulating tumor cell migration place the enzyme among the potential targets of anti-metastatic therapy.

Functional role of inositol-1,4,5-trisphosphate-3-kinase-A for motility of malignant transformed cells.

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.

Tubulin Tyrosine Ligase Like 4 (TTLL4) overexpression in breast cancer cells is associated with brain metastasis and alters exosome biogenesis.

BACKGROUND: The survival rate is poor in breast cancer patients with brain metastases. Thus, new concepts for therapeutic approaches are required. During metastasis, the cytoskeleton of cancer cells is highly dynamic and therefore cytoskeleton-associated proteins are interesting targets for tumour therapy. METHODS: Screening for genes showing a significant correlation with brain metastasis formation was performed based on microarray data from breast cancer patients with long-term follow up information. Validation of the most interesting target was performed by MTT-, Scratch- and Transwell-assay. In addition, intracellular trafficking was analyzed by live-cell imaging for secretory vesicles, early endosomes and multiple vesicular bodies (MVB) generating extracellular vesicles (EVs). EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western blotting, mass spectrometry, and ingenuity pathway analysis (IPA). Effect of EVs on the blood-brain-barrier (BBB) was examined by incubating endothelial cells of the BBB (hCMEC/D3) with EVs, and permeability as well as adhesion of breast cancer cells were analyzed. Clinical data of a breast cancer cohort was evaluated by χ2-tests, Kaplan-Meier-Analysis, and log-rank tests while for experimental data Student's T-test was performed. RESULTS: Among those genes exhibiting a significant association with cerebral metastasis development, the only gene coding for a cytoskeleton-associated protein was Tubulin Tyrosine Ligase Like 4 (TTLL4). Overexpression of TTLL4 (TTLL4plus) in MDA-MB231 and MDA-MB468 breast cancer cells (TTLL4plus cells) significantly increased polyglutamylation of ß-tubulin. Moreover, trafficking of secretory vesicles and MVBs was increased in TTLL4plus cells. EVs derived from TTLL4plus cells promote adhesion of MDA-MB231 and MDA-MB468 cells to hCMEC/D3 cells and increase permeability of hCMEC/D3 cell layer. CONCLUSIONS: These data suggest that TTLL4-mediated microtubule polyglutamylation alters exosome homeostasis by regulating trafficking of MVBs. The TTLL4plus-derived EVs may provide a pre-metastatic niche for breast cancer cells by manipulating endothelial cells of the BBB.

Physiological relevance of the neuronal isoform of inositol-1,4,5-trisphosphate 3-kinases in mice.

Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) is the neuronal isoform of ITPKs and exhibits both actin bundling and InsP3kinase activity. In addition to neurons, ITPKA is ectopically expressed in tumor cells, where its oncogenic activity increases tumor cell malignancy. In order to analyze the physiological relevance of ITPKA, here we performed a broad phenotypic screening of itpka deficient mice. Our data show that among the neurobehavioral tests analyzed, itpka deficient mice reacted faster to a hotplate, prepulse inhibition was impaired and the accelerating rotarod test showed decreased latency of itpka deficient mice to fall. These data indicate that ITPKA is involved in the regulation of nociceptive pathways, sensorimotor gating and motor learning. Analysis of extracerebral functions in control and itpka deficient mice revealed significantly reduced glucose, lactate, and triglyceride plasma concentrations in itpka deficient mice. Based on this finding, expression of ITPKA was analyzed in extracerebral tissues and the highest level was found in the small intestine. However, functional studies on CaCo-2 control and ITPKA depleted cells showed that glucose, as well as triglyceride uptake, were not significantly different between the cell lines. Altogether, these data show that ITPKA exhibits distinct functions in the central nervous system and reveal an involvement of ITPKA in energy metabolism.

Ins(1,4,5)P3 3-kinase-A overexpression induces cytoskeletal reorganization via a kinase-independent mechanism.

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.

Smooth Muscle-Alpha Actin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting Rac1 Activity.

Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration.

Inositol-1,4,5-trisphosphate-3-kinase-A controls morphology of hippocampal dendritic spines.

Long-lasting synaptic plasticity is often accompanied by morphological changes as well as formation and/or loss of dendritic spines. Since the spine cytoskeleton mainly consists of actin filaments, morphological changes are primarily controlled by actin binding proteins (ABPs). Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) is a neuron-specific, actin bundling protein concentrated at dendritic spines. Here, we demonstrate that ITPKA depletion in mice increases the number of hippocampal spine-synapses while reducing average spine length. By employing actin to ABP ratios similar to those occurring at post synaptic densities, in addition to cross-linking actin filaments, ITPKA strongly inhibits Arp2/3-complex induced actin filament branching by displacing the complex from F-actin. In summary, our data show that in vivo ITPKA negatively regulates formation and/or maintenance of synaptic contacts in the mammalian brain. On the molecular level this effect appears to result from the ITPKA-mediated inhibition of Arp2/3-complex F-actin branching activity.

The catalytic domain of inositol-1,4,5-trisphosphate 3-kinase-a contributes to ITPKA-induced modulation of F-actin.

Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process.

Malignant H1299 tumour cells preferentially internalize iron-bound inositol hexakisphosphate.

In colon enterocytes and in well-differentiated colon cancer CaCo-2 cells, InsP6 (inositol hexakisphosphate) inhibits iron uptake by forming extracellular insoluble iron/InsP6 complexes. In this study, we confirmed that CaCo-2 cells are not able to take up iron/InsP6 but, interestingly, found that the cells are able to internalize metal-free and Cr3+-bound InsP6. Thus, the inability of CaCo-2 cells to take up iron/InsP6 complexes seems to be due to the iron-bound state of InsP6. Since recently we demonstrated that the highly malignant bronchial carcinoma H1299 cells internalize and process InsP6, we examined whether these cells may be able to take up iron/InsP6 complexes. Indeed, we found that InsP6 dose-dependently increased uptake of iron and demonstrated that in the iron-bound state InsP6 is more effectively internalized than in the metal-free or Cr3+-bound state, indicating that H1299 cells preferentially take up iron/InsP6 complexes. Electron microscope and cell fraction assays indicate that after uptake H1299 cells mainly stored InsP6/iron in lysosomes as large aggregates, of which about 10% have been released to the cytosol. However, this InsP6-mediated iron transport had no significant effects on cell viability. This result together with our finding that the well-differentiated CaCo-2 cells did not, but the malignant H1299 cells preferentially took up iron/InsP6, may offer the possibility to selectively transport cytotoxic substances into tumour cells.

Inositol-1,4,5-trisphosphate 3-kinase A regulates dendritic morphology and shapes synaptic Ca2+ transients.

Inositol-1,4,5-trisphosphate 3-kinase-A (itpka) accumulates in dendritic spines and seems to be critically involved in synaptic plasticity. The protein possesses two functional activities: it phosphorylates inositol-1,4,5-trisphosphate (Ins(1,4,5)P(3)) and regulates actin dynamics by its F-actin bundling activity. To assess the relevance of these activities for neuronal physiology, we examined the effects of altered itpka levels on cell morphology, Ins(1,4,5)P(3) metabolism and dendritic Ca(2+) signaling in hippocampal neurons. Overexpression of itpka increased the number of dendritic protrusions by 71% in immature primary neurons. In mature neurons, however, the effect of itpka overexpression on formation of dendritic spines was weaker and depletion of itpka did not alter spine density and synaptic contacts. In synaptosomes of mature neurons itpka loss resulted in decreased duration of Ins(1,4,5)P(3) signals and shorter Ins(1,4,5)P(3)-dependent Ca(2+) transients. At synapses of itpka deficient neurons the levels of Ins(1,4,5)P(3)-5-phosphatase (inpp5a) and sarcoplasmic/endoplasmic reticulum calcium ATPase pump-2b (serca2b) were increased, indicating that decreased duration of Ins(1,4,5)P(3) and Ca(2+) signals results from compensatory up-regulation of these proteins. Taken together, our data suggest a dual role for itpka. In developing neurons itpka has a morphogenic effect on dendrites, while the kinase appears to play a key role in shaping Ca(2+) transients at mature synapses.