The epigenetic factor KDM2B regulates cell adhesion, small rho GTPases, actin cytoskeleton and migration in prostate cancer cells.

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.

The prognostic value of JUNB-positive CTCs in metastatic breast cancer: from bioinformatics to phenotypic characterization.

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.

Upregulation of Orai1 and STIM1 expression as well as store-operated Ca2+ entry in ovary carcinoma cells by placental growth factor.

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.

Taurolidine Sensitivity of Eryptosis, the Suicidal Erythrocyte Death.

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.

Stimulation of Eryptosis, the Suicidal Erythrocyte Death, by Costunolide.

BACKGROUND/AIMS: The sesquiterpene lactone Costunolide is effective against various disorders including inflammation and malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Mechanisms involved include altered function of transcription factors and mitochondria. Erythrocytes lack nuclei and mitochondria but are - in analogy to apoptosis of nucleated cells - able to enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Costunolide induces eryptosis and, if so, to shed light on the mechanisms involved. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from 2',7'-dichlorodihydrofluorescein (DCF)-dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to Costunolide (15 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence, DCF-fluorescence, and ceramide abundance. The effect of Costunolide on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. CONCLUSION: Costunolide triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by oxidative stress and ceramide formation.

The Epigenetic Factor KDM2B Regulates EMT and Small GTPases in Colon Tumor Cells.

BACKGROUND/AIMS: The epigenetic factor KDM2B is a histone demethylase expressed in various tumors. Recently, we have shown that KDM2B regulates actin cytoskeleton organization, small Rho GTPases signaling, cell-cell adhesion and migration of prostate tumor cells. In the present study, we addressed its role in regulating EMT and small GTPases expression in colon tumor cells. METHODS: We used RT-PCR for the transcriptional analysis of various genes, Western blotting for the assessment of protein expression and immunofluorescence microscopy for visualization of fluorescently labeled proteins. RESULTS: We report here that KDM2B regulates EZH2 and BMI1 in HCT116 colon tumor cells. Knockdown of this epigenetic factor induced potent up-regulation of the protein levels of the epithelial markers E-cadherin and ZO-1, while the mesenchymal marker N-cadherin was downregulated. On the other hand, KDM2B overexpression downregulated the levels of both epithelial markers and upregulated the mesenchymal marker, suggesting control of EMT by KDM2B. In addition, RhoA, RhoB and RhoC protein levels diminished upon KDM2B-knockdown, while all three small GTPases became upregulated in KDM2B-overexpressing HCT116 cell clones. Interestingly, Rac1 GTPase level increased upon KDM2B-knockdown and diminished in KDM2B-overexpressing HCT116 colon tumor- and DU-145 prostate cancer cells. CONCLUSIONS: These results establish a clear functional role of the epigenetic factor KDM2B in the regulation of EMT and small-GTPases expression in colon tumor cells and further support the recently postulated oncogenic role of this histone demethylase in various tumors.

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

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.

Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells.

BACKGROUND/AIMS: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. METHODS: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements. RESULTS: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. CONCLUSION: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development.

The histone demethylase KDM2B activates FAK and PI3K that control tumor cell motility.

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.

CXCR4 and JUNB double-positive disseminated tumor cells are detected frequently in breast cancer patients at primary diagnosis.

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.

Decreased Na+/K+ ATPase Expression and Depolarized Cell Membrane in Neurons Differentiated from Chorea-Acanthocytosis Patients.

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.

Serum- and glucocorticoid-inducible kinase 1 and the response to cell stress.

Expression of the serum- and glucocorticoid-inducible kinase 1 (SGK1) is up-regulated by several types of cell stress, such as ischemia, radiation and hyperosmotic shock. The SGK1 protein is activated by a signaling cascade involving phosphatidylinositide-3-kinase (PI3K), 3-phosphoinositide-dependent kinase 1 (PDK1) and mammalian target of rapamycin (mTOR). SGK1 up-regulates Na+/K+-ATPase, a variety of carriers including Na+-,K+-,2Cl-- cotransporter (NKCC), NaCl cotransporter (NCC), Na+/H+ exchangers, diverse amino acid transporters and several glucose carriers such as Na+-coupled glucose transporter SGLT1. SGK1 further up-regulates a large number of ion channels including epithelial Na+ channel ENaC, voltagegated Na+ channel SCN5A, Ca2+ release-activated Ca2+ channel (ORAI1) with its stimulator STIM1, epithelial Ca2+ channels TRPV5 and TRPV6 and diverse K+ channels. Furthermore, SGK1 influences transcription factors such as nuclear factor kappa-B (NF-κB), p53 tumor suppressor protein, cAMP responsive element-binding protein (CREB), activator protein-1 (AP-1) and forkhead box O3 protein (FOXO3a). Thus, SGK1 supports cellular glucose uptake and glycolysis, angiogenesis, cell survival, cell migration, and wound healing. Presumably as last line of defense against tissue injury, SGK1 fosters tissue fibrosis and tissue calcification replacing energy consuming cells.