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Loss of function mutations of the chorein-encoding gene VPS13A lead to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with accelerated suicidal neuronal cell death, which could be reversed by lithium. Chorein upregulates the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the Na+/K+-ATPase, a pump required for cell survival. To explore whether chorein-deficiency affects Na+/K+ pump capacity, cortical neurons were differentiated from iPSCs generated from fibroblasts of ChAc patients and healthy volunteers. Na+/K+ pump capacity was estimated from K+-induced whole cell outward current (pump capacity). As a result, the pump capacity was completely abolished in the presence of Na+/K+ pump-inhibitor ouabain (100 µM), was significantly smaller in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (24 hours 2 mM). The effect of lithium was reversed by SGK1-inhibitor GSK650394 (24 h 10 µM). Transmembrane potential (Vm) was significantly less negative in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (2 mM, 24 hours). The effect of lithium on Vm was virtually abrogated by ouabain. Na+/K+ α1-subunit transcript levels and protein abundance were significantly lower in ChAc neurons than in control neurons, an effect reversed by lithium treatment (2 mM, 24 hours). In conclusion, consequences of chorein deficiency in ChAc include impaired Na+/K+ pump capacity.

Recent studies revealed that the histone demethylase KDM2B regulates the epithelial markers E-Cadherin and ZO-1, the RhoA/B/C-small-GTPases and actin cytoskeleton organization, in DU-145 prostate- and HCT-116 colon-tumor cells. Here we addressed the role of KDM2B in the activation of Focal Adhesion Kinase (FAK)-signaling and its involvement in regulating tumor cell motility. We used RT-PCR for gene transcriptional analysis, Western blotting for the assessment of protein expression and activity and wound-healing assay for the study of cell migration. KDM2B overexpression or silencing controls the activity of FAK in DU-145 prostate- and HCT-116 colon-tumor cells without affecting gene transcription and protein expression of this kinase. Upon KDM2B overexpression in DU-145 cells, significantly enhanced migration was observed, which was abolished in cells pretreated by the specific phosphoinositide-3 kinase (PI3 K) inhibitor LY294002, implying involvement of FAK/PI3 K signaling in the migration process. In line with this, the p85-PI3 K-subunit was downregulated upon knockdown of KDM2B in DU-145 cells, while the opposite effect became evident in KDM2B-overexpressing cells. These results revealed a novel functional role of KDM2B in regulating the activation of the FAK/PI3 K signaling in prostate cancer cells that participates in the control of cell motility.

Placental growth factor (PlGF) is produced by tumor cells and stimulates tumor growth and metastasis in part by upregulation of hypoxia inducible factor HIF1α. Orchestration of tumor cell proliferation and migration involves oscillations of cytosolic Ca2+ activity ([Ca2+]i). The [Ca2+]i oscillations could be accomplished by triggering of intracellular Ca2+ release followed by store-operated Ca2+-entry (SOCE). Mechanisms accomplishing SOCE include the pore-forming ion channel unit Orai1 and its regulator STIM1. The present study explored whether PlGF influences the expression of Orai1 and STIM1, as well as SOCE and whether this effect impacts on HIF1α expression. To this end, ovary carcinoma cells were cultured for 24 h without and with PlGF (10 ng/ml). Orai1, STIM1 and HIF1α transcript levels were quantified utilizing RT-PCR and Orai1, STIM1 and HIF1α protein levels by Western blotting. [Ca2+]i was estimated from Fura-2-fluorescence and SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with extracellular Ca2+ removal and sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 μM). As a result, exposure of ovary carcinoma cells to PlGF was followed by a significant increase of Orai1 as well as STIM1 transcript and protein levels. PlGF significantly increased store-operated Ca2+-entry following re-addition of extracellular Ca2+, an effect virtually abrogated by Orai1 inhibitor MRS1845 (10 μM). PlGF further increased HIF1α transcript and protein levels, an effect again significantly blunted by MRS1845 (10 μM). In conclusion, PlGF upregulates expression of both, Orai1 and STIM1 thus enhancing store-operated Ca2+-entry with subsequent upregulation of HIF1α.

Background/Aims: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. Methods: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. Results: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). Conclusion: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

Background Circulating tumor cells (CTCs) are important for metastatic dissemination of cancer. They can provide useful information, regarding biological features and tumor heterogeneity; however, their detection and characterization are difficult due to their limited number in the bloodstream and their mesenchymal characteristics. Therefore, new biomarkers are needed to address these questions. Methods Bioinformatics functional enrichment analysis revealed a subgroup of 24 genes, potentially overexpressed in CTCs. Among these genes, the chemokine receptor CXCR4 plays a central role. After prioritization according to the CXCR4 corresponding pathways, five molecules (JUNB, YWHAB, TYROBP, NFYA, and PRDX1) were selected for further analysis in biological samples. The SKBR3, MDA-MB231, and MCF7 cell lines, as well as PBMCs from normal (n = 10) blood donors, were used as controls to define the expression pattern of all the examined molecules. Consequently, 100 previously untreated metastatic breast cancer (mBC) patients (n = 100) were analyzed using the following combinations of antibodies: CK (cytokeratin)/CXCR4/JUNB, CK/NFYA/ΥWHΑΒ (14-3-3), and CK/TYROBP/PRDX1. A threshold value for every molecule was considered the mean expression in normal PBMCs. Results Quantification of CXCR4 revealed overexpression of the receptor in SKBR3 and in CTCs, following the subsequent scale (SKBR3>CTCs>Hela>MCF7>MDA-MB231). JUNB was also overexpressed in CTCs (SKBR3>CTCs>MCF7>MDA-MB231>Hela). According to the defined threshold for each molecule, CXCR4-positive CTCs were identified in 90% of the patients with detectable tumor cells in their blood. In addition, 65%, 75%, 14.3%, and 12.5% of the patients harbored JUNB-, TYROBP-, NFYA-, and PRDX-positive CTCs, respectively. Conversely, none of the patients revealed YWHAB-positive CTCs. Interestingly, JUNB expression in CTCs was phenotypically and statistically enhanced compared to patients’ blood cells (p = 0.002) providing a possible new biomarker for CTCs. Furthermore, the detection of JUNB-positive CTCs in patients was associated with poorer PFS (p = 0.015) and OS (p = 0.002). Moreover, JUNB staining of 11 primary and 4 metastatic tumors from the same cohort of patients revealed a dramatic increase of JUNB expression in metastasis. Conclusions CXCR4, JUNB, and TYROBP were overexpressed in CTCs, but only the expression of JUNB was associated with poor prognosis, providing a new biomarker and a potential therapeutic target for the elimination of CTCs. Electronic supplementary material The online version of this article (10.1186/s13058-019-1166-4) contains supplementary material, which is available to authorized users.

Loss of function mutations of the chorein-encoding gene VPS13A lead to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with accelerated suicidal neuronal cell death, which could be reversed by lithium. Chorein upregulates the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the Na+/K+-ATPase, a pump required for cell survival. To explore whether chorein-deficiency affects Na+/K+ pump capacity, cortical neurons were differentiated from iPSCs generated from fibroblasts of ChAc patients and healthy volunteers. Na+/K+ pump capacity was estimated from K+-induced whole cell outward current (pump capacity). As a result, the pump capacity was completely abolished in the presence of Na+/K+ pump-inhibitor ouabain (100 µM), was significantly smaller in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (24 hours 2 mM). The effect of lithium was reversed by SGK1-inhibitor GSK650394 (24 h 10 µM). Transmembrane potential (Vm) was significantly less negative in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (2 mM, 24 hours). The effect of lithium on Vm was virtually abrogated by ouabain. Na+/K+ α1-subunit transcript levels and protein abundance were significantly lower in ChAc neurons than in control neurons, an effect reversed by lithium treatment (2 mM, 24 hours). In conclusion, consequences of chorein deficiency in ChAc include impaired Na+/K+ pump capacity.

Placental growth factor (PlGF) is produced by tumor cells and stimulates tumor growth and metastasis in part by upregulation of hypoxia inducible factor HIF1α. Orchestration of tumor cell proliferation and migration involves oscillations of cytosolic Ca

Background/Aims: The taurine derivative Taurolidine is effective against diverse bacteria and tumor growth. In the treatment of cancer, the substance is effective in part by triggering suicidal death or apoptosis of tumor cells. The Taurolidine-induced apoptosis involves mitochondria. Erythrocytes lack mitochondria but are nevertheless able to enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explores, whether Taurolidine induces eryptosis and, if so, which cellular mechanisms are involved. Methods: Phosphatidylserine exposure at the cell surface was estimated using annexin-V-binding, cell volume using forward scatter, [Ca2+]i using Fluo3-fuorescence, reactive oxygen species (ROS) formation using 2’,7’-dichlorodihydrofuorescein (DCF)-dependent fluorescence, and ceramide abundance using specific antibodies. Results: A 48 hours exposure of human erythrocytes to Taurolidine (60 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence and ceramide abundance, but not DCF-fluorescence. The effect of Taurolidine on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Taurolidine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by increase of ceramide abundance.

Background Circulating tumor cells (CTCs) are the major players in the metastatic process. A potential mechanism of cell migration and invasion is the formation of microtentacles in tumor cells. These structures are supported by α-tubulin (TUB), detyrosinated α-tubulin (GLU), and vimentin (VIM). In the current study, we evaluated the expression of those cytoskeletal proteins in CTCs. Methods Forty patients with breast cancer (BC) (16 early and 24 metastatic) were enrolled in the study. CTCs were isolated using the ISET platform and stained with the following combinations of antibodies: pancytokeratin (CK)/VIM/TUB and CK/VIM/GLU. Samples were analyzed with the ARIOL platform and confocal laser scanning microscopy. Results Fluorescence quantification revealed that the ratios CK/TUB, CK/VIM, and CK/GLU were statistically increased in MCF7 compared with more aggressive cell lines (SKBR3 and MDA-MB-231). In addition, all of these ratios were statistically increased in MCF7 cells compared with metastatic BC patients’ CTCs (p = 0.0001, p = 0.0001, and p = 0.003, respectively). Interestingly, intercellular connections among CTCs and between CTCs and blood cells through cytoskeleton bridges were revealed, whereas microtentacles were increased in patients with CTC clusters. These intercellular connections were supported by TUB, VIM, and GLU. Quantification of the examined molecules revealed that the median intensity of TUB, GLU, and VIM was significantly increased in patients with metastatic BC compared with those with early disease (TUB, 62.27 vs 11.5, p = 0.0001; GLU, 6.99 vs 5.29, p = 0.029; and VIM, 8.24 vs 5.38, p = 0.0001, respectively). Conclusions CTCs from patients with BC aggregate to each other and to blood cells through cytoskeletal protrusions, supported by VIM, TUB, and GLU. Quantification of these molecules could potentially identify CTCs related to more aggressive disease. Electronic supplementary material The online version of this article (10.1186/s13058-018-0993-z) contains supplementary material, which is available to authorized users.

Introduction: The presence of Disseminated (DTCs) and Circulating Tumor Cells (CTCs) in the bone marrow (BM) and the blood of breast cancer (BC) patients are poor prognostic factors. We have recently shown that CTCs derived from metastatic BC patients, overexpressed CXCR4 and JUNB and this expression was related to clinical outcome. In this study we applied the same methodology to BM of non-metastatic BC patients at primary diagnosis, before the onset of therapy. Methods: Bilateral BM (10 ml) aspirations were assessed for the detection of DTCs by a Ficoll density gradient centrifugation and subsequent centrifugation of the BM cells onto glass slides [1x106 mononuclear cells (MNCs) per slide]. Triple staining experiments with panytokeratin/CXCR4/JUNB antibodies were performed. An expression pattern of the examined proteins was created, using three different BC cell lines (SKBR3, MDA-MB231, and MCF7). Consequently, 10 BM aspirations from BC patients were analyzed. Mean intensity for each examined molecule was calculated automatically with the ARIOL system. Results: Quantification of CXCR4 among BC cell lines revealed that CXCR4 expression followed the subsequent hierarchy SKBR3>MCF7>MDA-MB231 with higher expression in SKBR3 and lower in MDA-MB 231 cells, respectively. Accordingly, the expression pattern of JUNB in cell lines was SKBR3>MCF7>MDA-MB231. Consequently, we analyzed 10 BM samples from primary BC patients. The (CXCR4+JUNB+CK+) phenotype was observed in 90% (9/10) of the samples. In addition, the (CXCR4-JUNB+CK+) phenotype was detected in 20% (2/10) of the patients. Interestingly, the (CXCR4+JUNB-CK+) phenotype was not observed in any of the patient's samples. Quantification of CXCR4 expression revealed that the mean intensity in DTCs (19.1±2) was statistically higher than in MCF7 (13±0.51; p=0.006) and MDA-MB231 (10.22±0.7; p= 0.0002) cell lines. It was also higher compared to normal donors' PBMCs (10.6±1.01; p=0.0004). In addition, the mean intensity of JUNB was statistically increased in DTCs (17.1±1.66) compared to MCF7 (8.9±0.56; p=0.00001) and MDA-MB231 (6.6±0.15; p= 0.00001) cells, respectively. Furthermore, it was increased compared to normal donors' PBMCs (5.2±0.15; p=0.0002). In two patients, we had available samples after treatment. In one patient, DTCs were eliminated after neoadjuvant therapy, for the other patient, DTCs were reduced from 10 to two DTCs. However, these cells were double positive for CXCR4 and JUNB. Conclusion: CXCR4 and JUNB were overexpressed in DTCs derived from primary BC patients, in agreement to previous findings in CTCs, implying an upregulation of the CXCR4 pathway in these cells. Quantification of this expression potentially defines a subgroup of patients with high expression of CXCR4 and JUNB that could benefit from targeted therapies. Citation Format: Galatea Kallergi, Oliver Hoffmann, Nefeli Zacharopoulou, Christos Stournaras, Vassilis Georgoulias, Sabine Kasmir-Bauer. Overexpression of CXCR4 and JUNB in disseminated tumor cells (DTCs) isolated from breast cancer patients before neoadjuvant treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5186.

Background:The chemokine receptor CXCR4 and the transcription factor JUNB, expressed on a variety of tumor cells, seem to play an important role in the metastatic process. Since disseminated tumor cells (DTCs) in the bone marrow (BM) have been associated with worse outcomes, we evaluated the expression of CXCR4 and JUNB in DTCs of primary, nonmetastatic breast cancer (BC) patients before the onset of any systemic treatment.Methods:Bilateral BM (10 ml) aspirations of 39 hormone receptor (HR)-positive, HER2-negative BC patients were assessed for the presence of DTCs using the following combination of antibodies: pan-cytokeratin (A45-B/B3)/CXCR4/JUNB. An expression pattern of the examined proteins was created using confocal laser scanning microscopy, Image J software and BC cell lines.Results:CXCR4 was overexpressed in cancer cells and DTCs, with the following hierarchy of expression: SKBR3 > MCF7 > DTCs > MDA-MB231. Accordingly, the expression pattern of JUNB was: DTCs > MDA-MB231 > SKBR3 > MCF7. The mean intensity of CXCR4 (6411 ± 334) and JUNB (27725.64 ± 470) in DTCs was statistically higher compared with BM hematopoietic cells (2009 ± 456, p = 0.001; and 11112.89 ± 545, p = 0.001, respectively). The (CXCR4+JUNB+CK+) phenotype was the most frequently detected [90% (35/39)], followed by the (CXCR4–JUNB+CK+) phenotype [36% (14/39)]. However, (CXCR4+JUNB–CK+) tumor cells were found in only 5% (3/39) of patients. Those patients harboring DTCs with the (CXCR4+JUNB+CK+) phenotype revealed lower overall survival (Cox regression: p = 0.023).Conclusions:(CXCR4+JUNB+CK+)-expressing DTCs, detected frequently in the BM of BC patients, seem to identify a subgroup of patients at higher risk for relapse that may be considered for close follow up.