Structural basis for subversion of host cell actin cytoskeleton during Salmonella infection.

Secreted bacterial type III secretion system (T3SS) proteins are essential for successful infection by many human pathogens. Both T3SS translocator SipC and effector SipA are critical for Salmonella infection by subversion of the host cell cytoskeleton, but the precise molecular interplay between them remains unknown. Here, using cryo-electron microscopy, we show that SipA binds along the F-actin grooves with a unique binding pattern. SipA stabilizes F-actin through charged interface residues and appears to prevent inorganic phosphate release through closure of the "back door" of adenosine 5'-triphosphate pocket. We also show that SipC enhances the binding of SipA to F-actin, thus demonstrating that a sequential presence of T3SS proteins in host cells is associated with a sequence of infection events-starting with actin nucleation, filament growth, and stabilization. Together, our data explain the coordinated interplay of a precisely tuned and highly effective mechanism during Salmonella infection and provide a blueprint for interfering with Salmonella effectors acting on actin.

Cryo-EM structures of actin binding proteins as tool for drug discovery.

Cellular actin dynamic is controlled by a plethora of actin binding proteins (ABPs), including actin nucleating, bundling, cross-linking, capping, and severing proteins. In this review, regulation of actin dynamics by ABPs will be introduced, and the role of the F-actin severing protein cofilin-1 and the F-actin bundling protein L-plastin in actin dynamics discussed in more detail. Since up-regulation of these proteins in different kinds of cancers is associated with malignant progression of cancer cells, we suggest the cryogenic electron microscopy (Cryo-EM) structure of F- actin with the respective ABP as template for in silico drug design to specifically disrupt the interaction of these ABPs with F-actin.

Ex Vivo Model of Neuroblastoma Plasticity.

Tumor plasticity is essential for adaptation to changing environmental conditions, in particular during the process of metastasis. In this study, we compared morphological and biochemical differences between LAN-1 neuroblastoma (NB) cells recovered from a subcutaneous xenograft primary tumor (PT) and the corresponding three generations of bone metastasis (BM I-III). Moreover, growth behavior, as well as the response to chemotherapy and immune cells were assessed. For this purpose, F-actin was stained, mRNA and protein expression assessed, and lactate secretion analyzed. Further, we measured adhesion to collagen I, the growth rate of spheroids in the presence and absence of vincristine, and the production of IL-6 by peripheral blood mononuclear cells (PBMCs) co-incubated with PT or BM I-III. Analysis of PT and the three BM generations revealed that their growth rate decreased from PT to BM III, and accordingly, PT cells reacted most sensitively to vincristine. In addition, morphology, adaption to hypoxic conditions, as well as transcriptomes showed strong differences between the cell lines. Moreover, BM I and BM II cells exhibited a significantly different ability to stimulate human immune cells compared to PT and BM III cells. Interestingly, the differences in immune cell stimulation corresponded to the expression level of the cancer-testis antigen MAGE-A3. In conclusion, our ex vivo model allows to analyze the adaption of tumor populations to different microenvironments and clearly demonstrates the strong alteration of tumor cell populations during this process.

The actin bundling activity of ITPKA mainly accounts for its migration-promoting effect in lung cancer cells.

Expression of Ins(1,4,5)P3-kinase-A (ITPKA), the neuronal isoform of Ins(1,4,5)P3-kinases, is up-regulated in many tumor types. In particular, in lung cancer cells this up-regulation is associated with bad prognosis and it has been shown that a high level of ITPKA increases migration and invasion of lung cancer cell lines. However, since ITPKA exhibits actin bundling and Ins(1,4,5)P3-kinase activity, it was not clear which of these activities account for ITPKA-promoted migration and invasion of cancer cells. To address this issue, we inhibited endogenous actin bundling activity of ITPKA in lung cancer H1299 cells by overexpressing the dominant negative mutant ITPKAL34P. Analysis of actin dynamics in filopodia as well as wound-healing migration revealed that ITPKAL34P inhibited both processes. Moreover, the formation of invasive protrusions into collagen I was strongly blocked in cells overexpressing ITPKAL34P. Furthermore, we found that ATP stimulation slightly but significantly (by 13%) increased migration of cells overexpressing ITPKA while under basal conditions up-regulation of ITPKA had no effect. In accordance with these results, overexpression of a catalytic inactive ITPKA mutant did not affect migration, and the Ins(1,4,5)P3-kinase-inhibitor GNF362 reversed the stimulating effect of ITPKA overexpression on migration. In summary, we demonstrate that under basal conditions the actin bundling activity controls ITPKA-facilitated migration and invasion and in presence of ATP the Ins(1,4,5)P3-kinase activity slightly enhances this effect.

Key Role of Hyaluronan Metabolism for the Development of Brain Metastases in Triple-Negative Breast Cancer.

Breast cancer (BC) is the second-most common cause of brain metastases (BM) and BCBM patients have a reduced quality of life and a poor prognosis. Hyaluronan (HA), and in particular the hyaluronidase Hyal-1, has been already linked to the development of BCBM, and therefore presents an interesting opportunity to develop new effective therapeutic options. HA metabolism was further discovered by the CRISPR/Cas9-mediated knockout of HYAL1 and the shRNA-mediated down-regulation of HA-receptor CD44 in the brain-seeking triple-negative breast cancer (TNBC) cell line MDA-MB-231-BR. Therefore, the impact of Hyal-1 on adhesion, disruption, and invasion through the brain endothelium, both in vitro and in vivo, was studied. Our analysis points out a key role of Hyal-1 and low-molecular-weight HA (LMW-HA) in the formation of a pericellular HA-coat in BC cells, which in turn promotes tumor cell adhesion, disruption, and migration through the brain endothelium in vitro as well as the extent of BM in vivo. CD44 knockdown in MDA-MB-231-BR significantly reduced the pericellular HA-coat on these cells, and, consequently, tumor cell adhesion and invasion through the brain endothelium. Thus, the interaction between Hyal-1-generated LMW-HA fragments and the HA-receptor CD44 might represent a potential target for future therapeutic options in BC patients with a high risk of cerebral metastases formation.

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.

Overexpression of Lin28A in neural progenitor cells in vivo does not lead to brain tumor formation but results in reduced spine density.

LIN28A overexpression has been identified in malignant brain tumors called embryonal tumors with multilayered rosettes (ETMR) but its specific role during brain development remains largely unknown. Radial glia cells of the ventricular zone (VZ) are proposed as a cell of origin for ETMR. We asked whether an overexpression of LIN28A in such cells might affect brain development or result in the formation of brain tumors.Constitutive overexpression of LIN28A in hGFAP-cre::lsl-Lin28A (GL) mice led to a transient increase of proliferation in the cortical VZ at embryonic stages but no postnatal brain tumor formation. Postnatally, GL mice displayed a pyramidal cell layer dispersion of the hippocampus and altered spine and dendrite morphology, including reduced dendritic spine densities in the hippocampus and cortex. GL mice displayed hyperkinetic activity and differential quantitative MS-based proteomics revealed altered time dependent molecular functions regarding mRNA processing and spine morphogenesis. Phosphoproteomic analyses indicated a downregulation of mTOR pathway modulated proteins such as Map1b being involved in microtubule dynamics.In conclusion, we show that Lin28A overexpression transiently increases proliferation of neural precursor cells but it is not sufficient to drive brain tumors in vivo. In contrast, Lin28A impacts on protein abundancy patterns related to spine morphogenesis and phosphorylation levels of proteins involved in microtubule dynamics, resulting in decreased spine densities of neurons in the hippocampus and cortex as well as in altered behavior. Our work provides new insights into the role of LIN28A for neuronal morphogenesis and development and may reveal future targets for treatment of ETMR patients.

Mechanism of BIP-4 mediated inhibition of InsP3Kinase-A.

Overexpression of the neuronal InsP3kinase-A increases malignancy of different tumor types. Since InsP3kinase-A highly selectively binds Ins(1,4,5)P3, small molecules competing with Ins(1,4,5)P3 provide a promising approach for the therapeutic targeting of InsP3kinase-A. Based on this consideration, we analyzed the binding mechanism of BIP-4 (2-[3,5-dimethyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-5, 8-dinitro-1H-benzo[de]isoquinoline-1,3(2H)-dione), a known competitive small-molecule inhibitor of Ins(1,4,5)P3. We tested a total of 80 BIP-4 related compounds in biochemical assays. The results of these experiments revealed that neither the nitrophenyl nor the benzisochinoline group inhibited InsP3kinase-A activity. Moreover, none of the BIP-4 related compounds competed for Ins(1,4,5)P3, demonstrating the high selectivity of BIP-4. To analyze the inhibition mechanism of BIP-4, mutagenesis experiments were performed. The results of these experiments suggest that the nitro groups attached to the benzisochinoline ring compete for binding of Ins(1,4,5)P3 while the nitrophenyl group is associated with amino acids of the ATP-binding pocket. Our results now offer the possibility to optimize BIP-4 to design specific InsP3Kinase-A inhibitors suitable for therapeutic targeting of the enzyme.

Oncogenic activity and cellular functionality of melanoma associated antigen A3.

Cancer testis antigen Melanoma associated antigen A3 (MAGE-A3) has been subject of research for many years. Being expressed in various tumor types and influencing proliferation, metastasis, and tumor pathogenicity, MAGE-A3 is an attractive target for cancer therapy, particularly because in healthy tissues, MAGE-A3 is only expressed in testes and placenta. MAGE-A3 acts as a cellular master regulator by stimulating E3 ubiquitin ligase tripartite motif-containing protein 28 (TRIM28), resulting in regulation of various cellular targets. These include tumor suppressor protein p53 and cellular energy sensor AMP-activated protein kinase (AMPK). The restricted expression of MAGE-A3 in tumor cells makes MAGE-A3 an attractive target for vaccine-based immune therapy. However, although phase I and phase II clinical trials involving MAGE-A3-specific immunotherapeutic interventions were promising, large phase III studies failed. This article gives an overview about the role of MAGE-A3 as a cellular master switch and discusses approaches to improve MAGE-A3-based immunotherapies.

DIAPH1 regulates chromosomal instability of cancer cells by controlling microtubule dynamics.

Chromosomal instability (CIN) is a hallmark of cancer, resulting from misalignment and missegregation of chromosomes during meta- and anaphase, due to non-precise regulation of spindle-MT dynamics. Diaphanous Related Formin 1 (DIAPH1) is an actin nucleator and also binds microtubule (MT) with high affinity. In this study, we analyzed the role of DIAPH1 in regulation of spindle MT-dynamics and CIN in HT29 and HCT-116 colorectal cancer (CRC) cells. Our data show that down-regulation of DIAPH1 in these cell lines decreased spindle-MT speed by 50 % and the fraction of cells with misaligned and missegregated chromosomes was significantly increased. Furthermore, in HCT-116 DIAPH1 depleted cells deviation of chromosome number was elevated and the number of cells with micronuclei and cytosolic DNA was increased in both DIAPH1-knock down cell lines. In line with these results, database analysis revealed a significant correlation with low DIAPH1 mRNA expression and aneuploidy. Thus, DIAPH1 is substantially involved in the control of CIN in CRC cells. Since in vitro, DIAPH1 directly increased MT-polymerization, we assume that DIAPH1 controls CIN by regulating spindle-MT dynamics.

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.

Modeling Spontaneous Bone Metastasis Formation of Solid Human Tumor Xenografts in Mice.

The majority of cancer-related deaths are due to hematogenous metastases, and the bone marrow (BM) represents one of the most frequent metastatic sites. To study BM metastasis formation in vivo, the most efficient approach is based on intracardiac injection of human tumor cells into immunodeficient mice. However, such a procedure circumvents the early steps of the metastatic cascade. Here we describe the development of xenograft mouse models (balb/c rag2-/- and severe combined immunodeficient (SCID)), in which BM metastases are spontaneously derived from subcutaneous (s.c.) primary tumors (PTs). As verified by histology, the described methodology including ex vivo bioluminescence imaging (BLI) even enabled the detection of micrometastases in the BM. Furthermore, we established sublines from xenograft primary tumors (PTs) and corresponding BM (BM) metastases using LAN-1 neuroblastoma xenografts as a first example. In vitro "metastasis" assays (viability, proliferation, transmigration, invasion, colony formation) partially indicated pro-metastatic features of the LAN-1-BM compared to the LAN-1-PT subline. Unexpectedly, after s.c. re-injection into mice, LAN-1-BM xenografts developed spontaneous BM metastases less frequently than LAN-1-PT xenografts. This study provides a novel methodologic approach for modelling the spontaneous metastatic cascade of human BM metastasis formation in mice. Moreover, our data indicate that putative bone-metastatic features get rapidly lost upon routine cell culture.

Characterization of the substrate specificity of the inositol 5-phosphatase SHIP1.

SHIP1 is an inositol 5-phosphatase which is well established for its tumour suppressor potential in leukaemia. Enzymatically, two SHIP1 substrates, PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4 have been identified to date. Additional substrates were found for the homologue SHIP2. In this study, we identified new inositol phosphate (InsP) substrates of SHIP1 by metal dye detection high-performance liquid chromatography and compared the substrate profiles of SHIP1 and SHIP2. We were able to verify Ins(1,3,4,5)P4 as a substrate of SHIP1 and interestingly found Ins(1,2,3,4,5)P5 and Ins(2,3,4,5)P4 to be preferably used as substrates and Ins(1,4,5,6)P4 and Ins(2,4,5,6)P4 to be weak substrates. All of those except Ins(2,3,4,5)P4 are also known substrates of SHIP2 indicating a possible exclusive role of Ins(2,3,4,5)P4 hydrolysis for SHIP1 but not SHIP2 function.

The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia.

Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.

The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domain.

In this study, we analyzed the functional role of the formin Drosophila Homologue of Diaphanous2 (Diaph2) in colorectal cancer cells. We show that stable down-regulation of Diaph2 expression in HT29 cells decreased chromosome alignment and the velocity of chromosome movement during M-phase, thus reducing the proliferation rate and colony formation. In interphase cells, Diaph2 was diffusely distributed in the cytosol, while in metaphase cells the protein was located to spindle microtubules (MTs). Diaph2-depletion increased the concentration of stable spindle MTs, showing that the formin is required to control spindle MT-dynamics. Our cellular data indicate that Diaph2-controls spindle MT-dynamics independent of Cdc42 activity and our in vitro results reveal that bacterially produced full-length (FL) Diaph2 strongly altered MT-dynamics in absence of Cdc42, where its actin-nucleating activity is auto-inhibited. FL-Diaph2 mediates a 10-fold increase in MT-polymerization compared to the Diaph2-FH2-domain. Interestingly, a Diaph2-mutant lacking the FH2-domain (ΔFH2) increased MT-polymerization to a similar extent as the FH2-domain, indicating the existence of a second MT-binding domain. However, in contrast to FL-Diaph2 and the FH2-domain, ΔFH2 did not alter the density of taxol-stabilized MTs. Thus, the FH2-domain and the second Diaph2-binding domain appear to control MT-dynamics by different mechanisms. In summary, our data indicate that Diaph2 controls M-phase progression under basal conditions by regulating spindle MT-dynamics. In addition, a region outside of the canonical MT-regulating FH2-domain is involved in Diaph2-mediated control of MT-dynamics.

New options of cancer treatment employing InsP6.

Many mechanistic studies have been performed to analyze the cellular functions of the highly phosphorylated molecule inositol hexakisphosphate (InsP6) in health and disease. While the physiological intracellular functions are well described, the mechanism of potential pharmacological effects on cancer cell proliferation is still controversial. There are numerous studies demonstrating that a high InsP6 concentration (≥75 µM) inhibits growth of cancer cells in vitro and in vivo. Thus, there is no doubt that InsP6 exhibits anticancer activity but the mechanism underlying the cellular effects of extracellular InsP6 on cancer cells is far from being understood. In addition, studies on the inhibitory effect of InsP6 on cancer progression in animal models ignore aspects of its bioavailability. Here, we review and critically discuss the uptake mechanism and the intracellular involvement in signaling pathways of InsP6 in cancer cells. We take into account the controversial findings on InsP6 plasma concentration, which is a critical aspect of pharmacological accessibility of InsP6 for cancer treatment. Further, we discuss novel findings with respect to the effect of InsP6 on normal and immune cells as well as on platelet aggregate size. Our goal is to stimulate further mechanistic studies into novel directions considering previously disregarded aspects of InsP6. Only when we fully understand the mechanism underlying the anticancer activity of InsP6 novel and more efficient treatment options can be developed.

Differential Proteome Analysis of Human Neuroblastoma Xenograft Primary Tumors and Matched Spontaneous Distant Metastases.

Metastasis formation is the major cause for cancer-related deaths and the underlying mechanisms remain poorly understood. In this study we describe spontaneous metastasis xenograft mouse models of human neuroblastoma used for unbiased identification of metastasis-related proteins by applying an infrared laser (IR) for sampling primary tumor and metastatic tissues, followed by mass spectrometric proteome analysis. IR aerosol samples were obtained from ovarian and liver metastases, which were indicated by bioluminescence imaging (BLI), and matched subcutaneous primary tumors. Corresponding histology proved the human origin of metastatic lesions. Ovarian metastases were commonly larger than liver metastases indicating differential outgrowth capacities. Among ~1,900 proteins identified at each of the three sites, 55 proteins were differentially regulated in ovarian metastases while 312 proteins were regulated in liver metastases. There was an overlap of 21 and 7 proteins up- and down-regulated at both metastatic sites, respectively, most of which were so far not related to metastasis such as LYPLA2, EIF4B, DPY30, LGALS7, PRPH, and NEFM. Moreover, we established in vitro sublines from primary tumor and metastases and demonstrate differences in cellular protrusions, migratory/invasive potential and glycosylation. Summarized, this work identified several novel putative drivers of metastasis formation that are tempting candidates for future functional studies.

Clinical relevance of cytoskeleton associated proteins for ovarian cancer.

PURPOSE: Ovarian cancer has a high mortality rate and up to now no reliable molecular prognostic biomarkers have been established. During malignant progression, the cytoskeleton is strongly altered. Hence we analyzed if expression of certain cytoskeleton-associated proteins is correlated with clinical outcome of ovarian cancer patients. METHODS: First, in silico analysis was performed using the cancer genome atlas (TCGA), the human expression atlas and Pubmed. Selected candidates were validated on 270 ovarian cancer patients by qRT-PCR and/or by western blotting. RESULTS: In silico analysis revealed that mRNAs of 214 cytoskeleton-associated proteins are detectable in ovarian cancer tissue. Among these, we selected 17 proteins that participate in cancer disease progression and cytoskeleton modulation: KIF14, KIF20A, KIF18A, ASPM, CEP55, DLGAP5, MAP9, EB1, KATNA1, DIAPH1, ANLN, SCIN, CCDC88A, FSCN1, GSN, VASP and CDC42. The first ten candidates interact with microtubules (MTs) and the others bind to actin filaments. Validation on clinical samples of ovarian cancer patients revealed that the expression levels of DIAPH1, EB1, KATNA1, KIF14 and KIF18A significantly correlated with clinical and histological parameters of ovarian cancer. High DIAPH1, EB1, KATNA1 and KIF14 protein levels were associated with increased overall survival (OAS) of ovarian cancer patients, while high DIAPH1 and EB1 protein levels were also associated with low differentiation of respective tumors (G2/3). Moreover, DIAPH1 was the only protein, whose expression significantly correlated with increased recurrence-free interval (RFI). CONCLUSION: Mainly the expression levels of the MT-associated proteins analyzed in this study, correlated with prolonged survival of ovarian cancer patients. From > 200 genes initially considered, 17 cytoskeletal proteins are involved in cancer progression according to the literature. Among these, four proteins significantly correlated with improved survival of ovarian cancer patients.

Effect of the actin- and calcium-regulating activities of ITPKB on the metastatic potential of lung cancer cells.

Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) exhibits oncogenic activity in lung cancer cells by regulating Ins(1,4,5)P3-mediated calcium release and cytoskeletal dynamics. Since, in normal cells, ITPKA is mainly expressed in the brain, it is an excellent target for selected therapy of lung cancer. However, ITPKB is strongly expressed in normal lung tissues, but is down-regulated in lung cancer cells by miR-375, assuming that ITPKB might have tumor suppressor activity. In addition, ITPKB binds to F-actin making it likely that, similar to ITPKA, it controls actin dynamics. Thus, the treatment of ITPKA-expressing lung cancer with ITPKA inhibitors simultaneously inhibiting ITPKB may counteract the therapy. Based on these considerations, we analyzed if ITPKB controls actin dynamics and if the protein reduces aggressive progression of lung cancer cells. We found that ITPKB bundled F-actin in cell-free systems. However, the stable expression of ITPKB in H1299 lung cancer cells, exhibiting very low endogenous ITPKB expression, had no significant effect on the actin structure. In addition, our data show that ITPKB negatively controls transmigration of H1299 cells in vitro by blocking Ins(1,4,5)P3-mediated calcium release. On the other hand, colony formation was stimulated by ITPKB, independent of Ins(1,4,5)P3-mediated calcium signals. However, dissemination of H1299 cells from the skin to the lung in NOD scid gamma mice was not significantly affected by ITPKB expression. In summary, ITPKB does not affect the cellular actin structure and does not suppress dissemination of human lung cancer cells in mice. Thus, our initial hypotheses that ITPKB exhibits tumor suppressor activity could not be supported.