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Early and accurate diagnosis of prostate cancer (PCa) is extremely important, as metastatic PCa remains hard to treat. EWI‐2, a member of the Ig protein subfamily, is known to inhibit PCa cell migration. In this study, we found that EWI‐2 localized on both the cell membrane and exosomes regulates the distribution of miR‐3934‐5p between cells and exosomes. Interestingly, we observed that EWI‐2 is localized not only on the plasma membrane but also on the nuclear envelope (nuclear membrane), where it regulates the nuclear translocation of signaling molecules and miRNA. Collectively, these functions of EWI‐2 found in lipid bilayers appear to regulate PCa cell metastasis through the epidermal growth factor receptor‐mitogen‐activated protein kinase‐extracellular‐signal‐regulated kinase (EGFR‐MAPK‐ERK) pathway. Our research provides new insights into the molecular function of EWI‐2 on PCa metastasis, and highlights EWI‐2 as a potential PCa biomarker., Our findings identify EWI‐2 as a potential prostate cancer (PCa) biomarker. EWI‐2 is involved in PCa metastasis through its localization on lipid bilayers where it facilitates nucleocytoplasmic shuttling of EGFR signaling molecules and also regulates microRNA sorting in exosomes. These molecular functions of EWI‐2 result in downstream activation of the epidermal growth factor receptor‐mitogen‐activated protein kinase‐extracellular‐signal‐regulated kinase (EGFR‐MAPK‐ERK) signaling pathway and promote PCa cell migration.

Through integration of multiple large database, two oncogenic signatures (YAP conserved and TGFβ‐up signatures) were commonly upregulated in the cancer cell lines with the mesenchymal properties. AXL, the expression of which is highly induced in EMT, contributes the doxorubicin resistance in EMT. Doxorubicin treatment induced YAP‐dependent gene response and promoted AXL expression coordinated with TGFβ‐SMAD4., The acquisition of chemoresistance remains a major cause of cancer mortality due to the limited accessibility of targeted or immune therapies. However, given that severe alterations of molecular features during epithelial‐to‐mesenchymal transition (EMT) lead to acquired chemoresistance, emerging studies have focused on identifying targetable drivers associated with acquired chemoresistance. Particularly, AXL, a key receptor tyrosine kinase that confers resistance against targets and chemotherapeutics, is highly expressed in mesenchymal cancer cells. However, the underlying mechanism of AXL induction in mesenchymal cancer cells is poorly understood. Our study revealed that the YAP signature, which was highly enriched in mesenchymal‐type lung cancer, was closely correlated to AXL expression in 181 lung cancer cell lines. Moreover, using isogenic lung cancer cell pairs, we also found that doxorubicin treatment induced YAP nuclear translocation in mesenchymal‐type lung cancer cells to induce AXL expression. Additionally, the concurrent activation of TGFβ signaling coordinated YAP‐dependent AXL expression through SMAD4. These data suggest that crosstalk between YAP and the TGFβ/SMAD axis upon treatment with chemotherapeutics might be a promising target to improve chemosensitivity in mesenchymal‐type lung cancer.

Huntington's disease (HD) is caused by a genetically mutated huntingtin (mHtt) protein with expanded polyQ stretch, which impairs cytosolic sequestration of the repressor element‐1 silencing transcription factor (REST), resulting in excessive nuclear REST and subsequent repression of neuronal genes. We recently demonstrated that REST undergoes extensive, context‐dependent alternative splicing, of which exon‐3 skipping (∆E3)—a common event in human and nonhuman primates—causes loss of a motif critical for REST nuclear targeting. This study aimed to determine whether ∆E3 can be targeted to reduce nuclear REST and rescue neuronal gene expression in mouse striatal‐derived, mHtt‐expressing STHdhQ111/Q111 cells—a well‐established cellular model of HD. We designed two morpholino antisense oligos (ASOs) targeting the splice sites of Rest E3 and examined their effects on ∆E3, nuclear Rest accumulation and Rest‐controlled gene expression in STHdhQ111/Q111 cells. We found that (1) the ASOs treatment significantly induced ∆E3, reduced nuclear Rest, and rescued transcription and/or mis‐splicing of specific neuronal genes (e.g. Syn1 and Stmn2) in STHdhQ111/Q111 cells; and (2) the ASOs‐induced transcriptional regulation was dependent on ∆E3 induction and mimicked by siRNA‐mediated knock‐down of Rest expression. Our findings demonstrate modulation of nuclear REST by ∆E3 and its potential as a new therapeutic target for HD and provide new insights into environmental regulation of genome function and pathogenesis of HD. As ∆E3 is modulated by cellular signalling and linked to various types of cancer, we anticipate that ∆E3 contributes to environmentally tuned REST function and may have a broad range of clinical implications.

Parabens owing to their many advantageous properties are widely applied in cosmetics, food products and pharmaceuticals. However, recent research results have shown that they possess the ability to accumulate in the human body and exert many adverse effects. In this study, the impact of methylparaben (MP) as the most frequently used preservative in cosmetics, on human dermal fibroblasts and collagen production was evaluated. In cells treated with 0.01, 0.03 and 0.05% MP a dose-dependent decrease in collagen biosynthesis was revealed, which was positively correlated with the activity of prolidase responsible for the recovery of proline. Consequently, the concentration of total collagen secreted into the medium was markedly diminished. A similar reduction in expression of the major skin collagen type I at both the protein and mRNA level as well as collagen type III and VI at the mRNA level was also detected. The decrease in the collagen level may result not only from the reduced synthesis but also increased degradation owing to MP-induced activation of pro-MMP-2 (72 kDa). The increase in activity of MMP-2 (66 kDa) was accompanied by a reduction in the inhibitory activity of TIMP-2. In addition, an inhibitory effect of MP on cell survival and proliferation was revealed in this study. The increased expression and nuclear translocation of caspase-3 as well as increased Bax and decreased Bcl-2 expression may suggest MP-induced cell apoptosis. In summary, we have provided new data on the adverse effects of methylparaben on human dermal fibroblasts and the main structural protein of the skin. Further studies on the mechanisms responsible for its action are in progress. Copyright © 2017 John Wiley & Sons, Ltd.

Deregulation of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases (RTKs) has recently been demonstrated to predominately promote survival and chemoresistance of cancer cells. Intramembrane proteolysis mediated by presenilin/gamma-secretase is known to regulate the homeostasis of some RTKs. In the present study, we demonstrate that AXL, but not TYRO3 or MERTK, is efficiently and sequentially cleaved by alpha-and g-secretases in various types of cancer cell lines. Proteolytic processing of AXL redirected signaling toward a secretase-mediated pathway, away from the classic, well-known, ligand-dependent canonical RTK signaling pathway. TheAXL intracellular domain cleavage product, but not full-length AXL, was further shown to translocate into the nucleus via a nuclear localization sequence that harbored a basic HRRKK motif. Of interest, we found that the g-secretase-uncleavable AXL mutant caused an elevated chemoresistance in non-small-cell lung cancer cells. Altogether, our findings suggest thatAXL can undergo sequential processing mediated by various proteases kept in a homeostatic balance. This newly discovered post-translational processing ofAXL may provide an explanation for the diverse functions of AXL, especially in the context of drug resistance in cancer cells.-Lu, Y., Wan, J., Yang, Z., Lei, X., Niu, Q., Jiang, L., Passtoors, W. M., Zang, A., Fraering, P. C., Wu, F. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells. FASEB J. 31, 1382-1397 (2017). www.fasebj.org

Introduction Mutations in the angiogenin (ANG) gene are known to be associated with both familial and sporadic amyotrophic lateral sclerosis (ALS). The majority of disease‐causing mutations of ANG result in loss of either ribonucleolytic activity, nuclear translocation activity or both. Methods We sequenced ANG gene from a total of 136 sporadic ALS patients and 112 healthy controls of Hungarian origin. To elucidate the role of the R33W mutation in the disease mechanism, computational, and functional analyses were performed. Results Mutation screening revealed a mutation located in the signal peptide (M‐24I) and two mutations that affect the mature protein (R33W, V103I). The R33W mutation, which has not been previously detected in ALS patients, affects the key amino acid of the nuclear translocation signal of the ANG protein. Molecular dynamics simulations suggested that the R33W mutation results in partial loss of ribonucleolytic activity and reduced nuclear translocation activity. The ribonucleolytic assay and nuclear translocation assay of the R33W ANG protein confirmed the molecular dynamics results. Conclusions In the Hungarian ALS population, the observed frequency of ANG mutations was 2.9%, which is higher than previously reported for sporadic cohorts. The evidence from computational and functional analyses support the deleterious effect of the novel R33W variant detected in this study.

Natural extracellular matrix (ECM) mostly has a fibrous structure that supports and mechanically interacts with local residing cells to guide their behaviors. The effect of ECM elasticity on cell behaviors has been extensively investigated, while less attention has been paid to the effect of matrix fiber-network plasticity at microscale, although plastic remodeling of fibrous matrix is a common phenomenon in fibrosis. Here, a significant decrease is found in plasticity of native fibrotic tissues, which is associated with an increase in matrix crosslinking. To explore the role of plasticity in fibrosis development, a set of 3D collagen nanofibrous matrix with constant modulus but tunable plasticity is constructed by adjusting the crosslinking degree. Using plasticity-controlled 3D culture models, it is demonstrated that the decrease of matrix plasticity promotes fibroblast activation and spreading. Further, a coarse-grained molecular dynamic model is developed to simulate the cell-matrix interaction at microscale. Combining with molecular experiments, it is revealed that the enhanced fibroblast activation is mediated through cytoskeletal tension and nuclear translocation of Yes-associated protein. Taken together, the results clarify the effects of crosslinking-induced plasticity changes of nanofibrous matrix on the development of fibrotic diseases and highlight plasticity as an important mechanical cue in understanding cell-matrix interactions.

Chondroitin sulfate proteoglycan 4 (CSPG4) has been identified as a highly promising target antigen for immunotherapy of triple-negative breast cancer (TNBC). TNBC represents a highly aggressive heterogeneous group of tumors lacking expression of estrogen, progesterone and human epidermal growth factor receptor 2. TNBC is particularly prevalent among young premenopausal women. No suitable targeted therapies are currently available and therefore, novel agents for the targeted elimination of TNBC are urgently needed. Here, we present a novel cytolytic fusion protein (CFP), designated alpha CSPG4(scFv)-MAP, that consists of a high affinity CSPG4-specific single-chain antibody fragment (scFv) genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. Our data indicate that alpha CSPG4(scFv)-MAP efficiently targets CSPG4(+) TNBC-derived cell lines MDA-MB-231 and Hs 578T and potently inhibits their growth with IC50 values of similar to 200 nM. Treatment with alpha CSPG(scFv)-MAP resulted in induction of the mitochondrial stress pathway by activation of caspase-9 as well as endonuclease G translocation to the nucleus, while induction of the caspase-3 apoptosis pathway was not detectable. Importantly, in vivo studies in mice bearing human breast cancer xenografts revealed efficient targeting to and accumulation of alpha CSPG4(scFv)-MAP at tumor sites resulting in prominent tumor regression. Taken together, this preclinical proof of concept study confirms the potential clinical value of alpha CSPG4(scFv)-MAP as a novel targeted approach for the elimination of CSPG4-positive TNBC.

Hydroxy-safflower yellow A (HSYA) is the major active component of safflower, a traditional Asia herbal medicine well known for its cardiovascular protective activities. The purpose of this study was to investigate the effect of HSYA on TNF-α-induced inflammatory responses in arterial endothelial cells (AECs) and to explore the mechanisms involved. The results showed that HSYA suppressed the up-regulation of ICAM-1 expression in TNF-α-stimulated AECs in a dose-dependent manner. High concentration (120 μM) HSYA significantly inhibited the TNF-α-induced adhesion of RAW264.7 cells to AECs. HSYA blocked the TNFR1-mediated phosphorylation and degradation of IκBα and also prevented the nuclear translocation of NF-κB p65. Moreover, HSYA reduced the cell surface level of TNFR1 and increased the content of sTNFR1 in the culture media. TNF-α processing inhibitor-0 (TAPI-0) prevented the HSYA inhibition of TNFR1-induced IκBα degradation, implying the occurrence of TNFR1 shedding. Furthermore, HSYA induced phosphorylation of TNF-α converting enzyme (TACE) at threonine 735, which is thought to be required for its activation. Conclusively, HSYA suppressed TNF-α-induced inflammatory responses in AECs, at least in part by inhibiting the TNFR1-mediated classical NF-κB pathway. TACE-mediated TNFR1 shedding can be involved in this effect. Our study provides new evidence for the antiinflammatory and anti-atherosclerotic effects of HSYA. Copyright © 2016 John Wiley & Sons, Ltd.

The Deubiquitinating enzyme, Cylindromatosis (CYLD), has been established as a crucial regulator of B-cells. The present study was addressed to identify the nature of CYLD-dependent RNomics in patients of pediatric age group with B-ALL. The study revealed the presence of a novel mutant CYLD of 55 kDa in these patients. The mutant CYLD displayed its ability to restrict the cells in G2 phase of cell cycle, down-regulate PLK-1 and block the nuclear translocation of BCL3. Based upon these results, we propose that this mutant CYLD has the capacity to act as a differential marker characteristic of B-cell lymphoblastic leukemia. Pediatr Blood Cancer 2015;62:1066-1069. © 2015 Wiley Periodicals, Inc.

Nuclear translocation of proteins is an essential aspect of normal cell function, and defects in this process have been detected in many disease-associated conditions. The detection and quantification of nuclear translocation was significantly boosted by the association of robotized microscopy with automated image analysis, a technology designated as high-content screening. Image-based high-content screening and analysis provides the means to systematically observe cellular translocation events in time and space in response to chemical or genetic perturbation at large scale. This approach yields powerful insights into the regulation of complex signaling networks independently of preconceived notions of mechanistic relationships. In this review, we briefly overview the different mechanisms involved in nucleocytoplasmic protein trafficking. In addition, we discuss high-content approaches used to interrogate the mechanistic and spatiotemporal dynamics of cellular signaling events using Forkhead box O (FOXO) proteins and the nuclear factor-κB (NF-κB) as important and clinically relevant examples.

Gonadotropin-releasing hormone (GnRH) regulates the synthesis and secretion of follicle-stimulating hormone (FSH) by stimulating the transcription of Fshβ gene. Our iTRAQ quantitative proteomics result showed that the abundance of α-actinin4 (ACTN4) increased in the nuclei of LβT2 cells upon GnRH induction. Using RNA interference, reverse transcription and real-time PCR, luciferase and transient transfection assays, we proved that ACTN4 is involved in the regulation of mouse Fshβ gene (mFshβ) transcription and its C-terminal calmodulin (CaM)-like domain is crucial for this process. Our study suggests that ACTN4 nuclear translocation mediates GnRH stimulation of mFshβ gene transcription.

Clusterin gene expression is potently induced in experimental models in which apoptosis is activated, such as rat prostate involution following castration. Nevertheless, its precise physiological role has not yet been established, and both anti-apoptotic and pro-apoptotic functions have been suggested for this gene. Clusterin expression level depends on cell proliferation state, and we recently showed that its over-expression inhibited cell cycle progression of SV40-immortalized human prostate epithelial cells PNT2 and PNT1a. Here we studied clusterin expression in PNT1a cells subjected to serum-starvation with the aim of defining clusterin early molecular changes following apoptosis induction. Under serum-starvation conditions, decreased growth rate, slow rounding-up of cells, cell detachment, and formation of apoptotic bodies indicative of anoikis (detachment-induced apoptosis) were preceded by significant downregulation of 70 kDa clusterin precursor and upregulation of 45-40 kDa isoforms. On the 8th day of serum-free culturing, only the higher molecular weight protein-band of about 45 kDa was clearly induced and accumulated in detached cells and apoptotic bodies in which PARP was activated. Anoikis was preceded by induction and transloction of a 45-kDa clusterin isoform to the nucleus. Thus, nuclear targeting of a specific 45-kDa isoform of clusterin appeared to be an early and specific molecular signal triggering anoikis-death. Considering also that clusterin is downregulated during prostate cancer onset and progression, and that its upregulation has inhibited DNA synthesis and cell cycle progression of immortalized human prostate epithelial cells, we suggest that clusterin might be a new anti-oncogene in the prostate.

Udgivelsesdato: 2003-Jul-10 We previously identified the Fas-associated factor FAF1 as an in vitro substrate of protein kinase CK2 and determined Ser289 and Ser291 as phosphorylation sites. Here we demonstrate that these two serine residues are the only sites phosphorylated by CK2 in vitro, and that at least one site is phosphorylated in vivo. Furthermore, we analyzed putative physiological functions of FAF1 phosphorylation. The ability of FAF1 to potentiate Fas-induced apoptosis is not influenced by the FAF1 phosphorylation status; however, the nuclear import of a phosphorylation-deficient FAF1 mutant was delayed in comparison to wild-type FAF1.

Malnutrition impairs host immunity, resulting in high mortality and morbidity, secondary to infections. Nuclear factor kappaB (NFkappaB) plays a critical role in host defense, but how quickly refeeding normalizes the impaired NFkappaB activity in peritoneal resident cells (PRCs) is unknown. Our aim was to investigate the effects of 1-day ad libitum refeeding on severe diet restriction-induced NFkappaB activity in PRCs.Mice received chow, 146 g/kg per day (ad libitum) or 36.5 g/kg per day (severe diet restriction), for 7 days. One-half the mice in the diet-restricted group were then fed ad libitum for 1 day (refeeding). PRCs were harvested by peritoneal lavage. After incubation with tumor necrosis factor-alpha (TNF-alpha), nuclear translocation of NFkappaB in PRCs was investigated using laser scanning cytometry.The main subpopulation of PRCs was macrophages in all groups. Mean fluorescence intensity over the nuclear area at 0 or 100 ng/mL of TNF-alpha was 16 +/- 2 or 31 +/- 8* in the ad libitum, 20 +/- 4 or 19 +/- 3 in the severe diet-restricted, and 20 +/- 4 or 30 +/- 5* in the refeeding group, respectively (*p.05 versus 0 ng/mL of TNF-alpha in each group versus 100 ng/mL of TNF-alpha in diet-restricted group). Cytoplasmic accumulation of NFkappaB was significantly increased after TNF-alpha stimulation in the refed group but not in the ad libitum group.The blunted NFkappaB activity in PRCs, after exposure to inflammatory stimuli, was restored after 1 day of refeeding, with increased accumulation of NFkappaB in the cytoplasm. Even brief nutritional replenishment in malnutrition may improve host defense by restoring NFkappaB activity and thereby improving macrophage functions.

The present work evaluates the effect of olive oil phenols on NF-κB activity in human gastric adenocarcinoma cells. The total phenol content was measured by the Folin Ciocalteu method, whereas the composition was assessed by LC-MS/MS analysis. Secoiridoids represented 71% and 83% of the Italian and Spanish extracts, respectively, phenol alcohols were in the range 9–13%. Ligustroside aglycone was the most abundant (37% and 46%, respectively, in the Italian and Spanish sample), and the concentration of flavonoids AP and LU was below 1%. Phenol extracts were assayed at 0.25–7.5 µg/mL, whereas single compounds were at 0.5–25 µ m. Both the extracts inhibited the NF-κB driven transcription in a concentration-dependent manner: IC50 for the Italian and the Spanish extract were 0.86 and 1.28 µg/mL, respectively. The IC50 for individual compounds ranged from 4.5 to 13 µ m. All the compounds under study inhibited nuclear translocation as well. The data suggest that consumption of extra-virgin olive oil may be beneficial for preventing the onset of gastric inflammation leading to more serious diseases. Copyright © 2012 John Wiley & Sons, Ltd.

Dehydroxymethylepoxyquinomicin (DHMEQ) is a low-m.w. compound that strongly inhibits NF-κB. DHMEQ directly binds to NF-κB subunits and thereby inhibits their nuclear translocation and DNA binding. ...

Genes differentially expressed in the subjective day and night in the rat suprachiasmatic nucleus (SCN) were surveyed by differential display. A gene homologous to human casein kinase 1ϵ (CK1ϵ) was isolated, which initially appeared to be expressed in the suprachiasmatic nucleus (SCN) in a circadian manner. We here describe the cDNA cloning of the rat CK1ϵ and characterization of the protein products. The rCK1ϵ is predominantly expressed in the brain including the SCN, binds and phosphorylates mPer1, mPer2, and mPer3 in vitro, and translocates mPer1 and mPer3, but not mPer2, to the cell nucleus depending on its kinase activity when coexpressed with these Per proteins in COS-7 cells.

In porcine coronary arteries, short-term treatment with resveratrol (RSVL) substantially inhibited MAPK activity (IC50=37 μM); and immunoblot analyses revealed consistent reduction in the phosphorylation of ERK-1/-2, JNK-1 and p38, at active sites. Endothelin-1 (ET-1), a primary antecedent in coronary heart diseases, enhanced MAPK activity, phosphorylation and nuclear translocation in a concentration-responsive but RSVL-sensitive manner. RSVL had no effect on basal or forskolin-stimulated cAMP levels, a known downregulator of the MAPK cascade. Likewise, inhibition of MAPK by RSVL was not reversed by the estrogen receptor blockers tamoxifen and ICI-182,780. Conversely, RSVL remarkably attenuated basal and ET-1-evoked protein tyrosine phosphorylation. Because MAPKs promote smooth muscle proliferation and contraction, their current inhibition may contribute to the putative protection by RSVL against coronary heart diseases. These effects apparently do not involve interaction with estrogen receptors.

Transcription of NFkB-dependent genes is regulated by the nuclear translocation and accumulation of IkBa. In the nucleus, IkBa suppresses transcription of NFkB-dependent pro-inflammatory and anti-a...

Heat shock protein 90 (hsp90) regulates the stability and function of several kinases, including IKB kinases (IKKs). We investigated the mechanisms by which hsp90 inhibitors prevent induction of NF...

Calcineurin regulates the dephosphorylation and nuclear translocation of the cardiac hypertophy transcription factor, Nuclear Factor of Activated T-cells (NFAT). O-linked beta-N-acetyl glucosamine ...