ARMS-NF-κB signaling regulates intracellular ROS to induce autophagy-associated cell death upon oxidative stress.

Ankyrin repeat-rich membrane spanning (ARMS) plays roles in neural development, neuropathies, and tumor formation. Such pleiotropic function of ARMS is often attributed to diverse ARMS-interacting molecules in different cell context. However, it might be achieved by ARMS' effect on global biological mediator like reactive oxygen species (ROS). We established ARMS-knockdown in melanoma cells (siARMS) and in Drosophila eyes (GMR>dARMS RNAi ) and challenged them with H2O2. Decreased ARMS in both systems compromises nuclear translocation of NF-κB and induces ROS, which in turn augments autophagy flux and confers susceptibility to H2O2-triggered autophagic cell death. Resuming NF-κB activity or reducing ROS by antioxidants in siARMS cells and GMR>dARMS RNAi fly decreases intracellular peroxides level concurrent with reduced autophagy and attenuated cell death. Conversely, blocking NF-κB activity in wild-type flies/melanoma enhances ROS and induces autophagy with cell death. We thus uncover intracellular ROS modulated by ARMS-NFκB signaling primes autophagy for autophagic cell death upon oxidative stress.

Histone Deacetylase Inhibitor, Trichostatin A, Synergistically Enhances Paclitaxel-Induced Cytotoxicity in Urothelial Carcinoma Cells by Suppressing the ERK Pathway.

Trichostatin A (TSA), an antifungal antibiotic derived from Streptomyces, inhibits mammalian histone deacetylases, and especially, selectively inhibits class I and II histone deacetylase (HDAC) families of enzymes. TSA reportedly elicits an antiproliferative response in multifarious tumors. This study investigated the antitumor effects of TSA alone and in combination with paclitaxel when applied to two high-grade urothelial carcinoma (UC) cell lines (BFTC-905 and BFTC-909). Fluorescence-activated cell sorting, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay were used to assess TSA's cytotoxicity and effects on apoptosis induction. TSA induced synergistic cytotoxicity, when combined with paclitaxel (combination index < 1), resulted in concomitant suppression of paclitaxel-induced activation of phospho-extracellular signal-regulated kinase (ERK) 1/2. A xenograft nude mouse model confirmed that TSA enhances the antitumor effects of paclitaxel. These findings demonstrate that the administration of TSA in combination with paclitaxel elicits a synergistic cytotoxic response. The results of this study indicate that the chemoresistance of UC could be circumvented by combining HDAC inhibitors to target the ERK pathway.

Correction to: Trichostatin A, a histone deacetylase inhibitor, induces synergistic cytotoxicity with chemotherapy via suppression of Raf/MEK/ERK pathway in urothelial carcinoma.

In Fig. 1b, upper part, the cell viability counts after treatment with cisplatin and TSA in T24 cells was by mistake a duplication of the image for NTUB1 on the left. In the corrected version of Fig. 1, the image was replaced appropriately.

Trichostatin A, a histone deacetylase inhibitor, induces synergistic cytotoxicity with chemotherapy via suppression of Raf/MEK/ERK pathway in urothelial carcinoma.

In this study, we aimed to investigate the antitumor effects of trichostatin A (TSA), an antifungal antibiotic that inhibits histone deacetylase (HDAC) family of enzymes, alone or in combination with anyone of the three chemotherapeutic agents (cisplatin, gemcitabine, and doxorubicin) for the treatment of human urothelial carcinoma (UC). Two high-grade human UC cell lines (T24 and NTUB1) were used. Cytotoxicity and apoptosis were assessed by MTT assay and flow cytometry, respectively. The expression of phospho-c-Raf, phospho-MEK1/2, and phospho-ERK1/2 was measured by western blotting. ERK siRNA knockdown and the specific MEK inhibitor U0126 were used to examine the role of Raf/MEK/ERK signaling pathway in combined cytotoxicity of TSA and chemotherapy. TSA co-treatment with any one of the three chemotherapeutic agents induced synergistic cytotoxicity (combination index < 1) and concomitantly suppressed chemotherapeutic drug-induced activation of Raf-MEK-ERK pathway. Combination of ERK siRNA knockdown and treatment with the specific MEK inhibitor (U0126) enhanced the cytotoxic effects of the chemotherapy on UC cells. These observations were confirmed in a xenograft nude mouse model. Moreover, activated Raf/MEK/ERK pathway was observed in human bladder UC specimens from patients with chemoresistant status. In conclusion, TSA elicits a synergistic cytotoxic response in combination with chemotherapy via targeting the Raf/MEK/ERK pathway. TSA elicits synergistic cytotoxic response in combination with three DNA-damaging drugs (cisplatin, gemcitabine, and doxorubicin). Activated Raf/MEK/ERK pathway is involved in chemoresistant mechanism of UC. Combining chemotherapeutic agents with HDAC inhibitor (TSA) or with targeting Raf/MEK/ERK pathway is promising to circumvent chemoresistance in UCs.

Activation of mTORC1 Signaling is Required for Timely Hair Follicle Regeneration from Radiation Injury.

Transit amplifying cells (TACs) are highly proliferative in nature and tend to be sensitive to ionizing radiation. Due to the abundance of TACs that support the elongation of hair shafts, growing hair follicles are highly sensitive to radiation injury. How hair follicles repair themselves after radiation injury is unclear. In this study, we observed that in 4 Gy irradiated mice, hair follicle dystrophy was induced with apoptosis-driven loss of hair matrix cells, which are the TACs that fuel hair growth. The dystrophy was repaired within 96 h without significant hair loss, indicating that a regenerative attempt successfully restored the TAC population to resume anagen growth. Soon after irradiation, mTORC1 signaling was activated in the TAC compartment and its activation was maintained until the regeneration process was completed. Inhibition of mTORC1 by rapamycin treatment increased radiation-induced cell apoptosis, reduced cell proliferation and delayed restoration of Wnt signaling in the hair matrix after radiation injury, leading to prolonged dystrophy and hair loss. These results demonstrate that mTORC1 signaling is activated after irradiation and is required for timely regeneration of the TAC pool of hair follicles, so that hair growth can resume after radiation injury.

Conditional Knockout of Breast Carcinoma Amplified Sequence 2 (BCAS2) in Mouse Forebrain Causes Dendritic Malformation via ß-catenin.

Breast carcinoma amplified sequence 2 (BCAS2) is a core component of the hPrP19 complex that controls RNA splicing. Here, we performed an exon array assay and showed that ß-catenin is a target of BCAS2 splicing regulation. The regulation of dendrite growth and morphology by ß-catenin is well documented. Therefore, we generated conditional knockout (cKO) mice to eliminate the BCAS2 expression in the forebrain to investigate the role of BCAS2 in dendrite growth. BCAS2 cKO mice showed a microcephaly-like phenotype with a reduced volume in the dentate gyrus (DG) and low levels of learning and memory, as evaluated using Morris water maze analysis and passive avoidance, respectively. Golgi staining revealed shorter dendrites, less dendritic complexity and decreased spine density in the DG of BCAS2 cKO mice. Moreover, the cKO mice displayed a short dendrite length in newborn neurons labeled by DCX, a marker of immature neurons, and BrdU incorporation. To further examine the mechanism underlying BCAS2-mediated dendritic malformation, we overexpressed ß-catenin in BCAS2-depleted primary neurons and found that the dendritic growth was restored. In summary, BCAS2 is an upstream regulator of ß-catenin gene expression and plays a role in dendrite growth at least partly through ß-catenin.

dBRWD3 Regulates Tissue Overgrowth and Ectopic Gene Expression Caused by Polycomb Group Mutations.

To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.

MLN4924 Synergistically Enhances Cisplatin-induced Cytotoxicity via JNK and Bcl-xL Pathways in Human Urothelial Carcinoma.

Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma. However, the response rate is only 40-65%. This study investigated the anti-tumor effect and underlying mechanisms of the combination of cisplatin and the NEDD8-activating enzyme inhibitor MLN4924 in human bladder urothelial carcinoma. The combination of cisplatin and MLN4924 exerted synergistic cytotoxicity on two high-grade bladder urothelial carcinoma cell lines, NTUB1 and T24 (combination index <1). MLN4924 also potentiated the cisplatin-induced apoptosis and activation of caspase-3 and -7, phospho-histone H2A.X and PARP. c-Jun N-terminal kinase (JNK) activation and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) were also observed during cisplatin and MLN4924 treatment. Inhibition of JNK activation partially restored cell viability and Bcl-xL expression. Bcl-xL overexpression also rescued cell viability. MLN4924 significantly potentiated cisplatin-induced tumor suppression in urothelial carcinoma xenograft mice. In summary, MLN4924 synergistically enhanced the anti-tumor effect of cisplatin via an increase in DNA damage, JNK activation and down-regulation of Bcl-xL in urothelial carcinoma cells. These findings provide a new therapeutic strategy for the treatment of bladder cancer.

Spindle-F Is the Central Mediator of Ik2 Kinase-Dependent Dendrite Pruning in Drosophila Sensory Neurons.

During development, certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact. How these neurons regulate pruning activity in the dendrites remains unknown. Here, we identify a coiled-coil protein Spindle-F (Spn-F) that is required for dendrite pruning in Drosophila sensory neurons. Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning. Spn-F exhibits a punctate pattern in larval neurons, whereas these Spn-F puncta become redistributed in pupal neurons, a step that is essential for dendrite pruning. The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association, and depends on the function of microtubule motor dynein complex. Spn-F is a key component to link Ik2 kinase to dynein motor complex, and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning. Our findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons.

BCAS2 Regulates Delta-Notch Signaling Activity through Delta Pre-mRNA Splicing in Drosophila Wing Development.

Previously, we showed that BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing. In this study, we provide strong evidence that BCAS2 regulates the activity of Delta-Notch signaling via Delta pre-mRNA splicing. Depletion of dBCAS2 reduces Delta mRNA expression and leads to accumulation of Delta pre-mRNA, resulting in diminished transcriptions of Delta-Notch signaling target genes, such as cut and E(spl)m8. Furthermore, ectopic expression of human BCAS2 (hBCAS2) and Drosophila BCAS2 (dBCAS2) in a dBCAS2-deprived fly can rescue dBCAS2 depletion-induced wing damage to the normal phenotypes. These rescued phenotypes are correlated with the restoration of Delta pre-mRNA splicing, which affects Delta-Notch signaling activity. Additionally, overexpression of Delta can rescue the wing deformation by deprivation of dBCAS2; and the depletion of dBCAS2 can restore the aberrant eye associated with Delta-overexpressing retinas; providing supporting evidence for the regulation of Delta-Notch signaling by dBCAS2. Taken together, dBCAS2 participates in Delta pre-mRNA splicing that affects the regulation of Delta-Notch signaling in Drosophila wing development.

MLN4924, a novel protein neddylation inhibitor, suppresses proliferation and migration of human urothelial carcinoma: In vitro and in vivo studies.

MLN4924, a small molecule inhibitor of NEDD8 activating enzyme (NAE), has been reported to elicit an anti-tumor effect on various malignancies. In this study, we investigated the anti-tumor effect of MLN4924 in human urothelial carcinoma (UC) in vitro and in vivo by using three human UC cell lines of various grading (T24, NTUB1 and RT4). The impact of MLN4924 on UC cells was determined by measuring viability (MTT), proliferation (BrdU incorporation), cell cycle progression (flow cytometry with propidium iodide staining) and apoptosis (flow cytometry with annexin V-FITC labeling). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration and invasion was analyzed by Transwell and wound healing assays. We also evaluated the effects of MLN4924 on tumor growth by a SCID xenograft mouse model. The data show that MLN4924 induced dose-dependent cytotoxicity, anti-proliferation, anti-migration, anti-invasion and apoptosis in human UC cells, accompanied by activations of Bad, phospho-histone H2A.X, caspase-3, 7 and PARP, decreased level of phospho-Bcl2, and caused cell cycle retardation at the G2M phase. Moreover, MLN4924 activated endoplasmic reticulum stress-related molecules (caspase-4, phospho-eIF2α, ATF-4 and CHOP) and other stress responses (JNK and c-Jun activations). Finally, we confirmed MLN4924 inhibited tumor growth in a UC xenograft mouse model with minimal general toxicity. We concluded that MLN4924 induces apoptosis and cell cycle arrest, as well as activation of cell stress responses in human UC. These findings imply MLN4924 provides a novel strategy for the treatment of UC.

MLN4924, a Novel NEDD8-activating enzyme inhibitor, exhibits antitumor activity and enhances cisplatin-induced cytotoxicity in human cervical carcinoma: in vitro and in vivo study.

MLN4924, an inhibitor of NEDD8 activating enzyme (NAE), has been reported to have activity against various malignancies. Here, we investigated the antitumor properties of MLN4924 and MLN4924 in combination with cisplatin on human cervical carcinoma (CC) in vitro and in vivo. Two human CC cell lines, ME-180 and HeLa, were used in this study. The cytotoxic effects of MLN4924 and/or cisplatin were measured by cell viability (MTT), proliferation (BrdU incorporation), apoptosis (flow cytometry with annexin V-FITC labeling), and the expression of cell apoptosis-related proteins (Western blotting). In vivo efficacy was determined in Nu/Nu nude mice with ME-180 and HeLa xenografts. The results showed that MLN4924 elicited viability inhibition, anti-proliferation and apoptosis in human CC cells, accompanied by activations of apoptosis-related molecules and Bid, Bcl-2 phosphorylation interruption, and interference with cell cycle regulators. Moreover, MLN4924 caused an endoplasmic reticulum stress response (caspase-4, ATF-4 and CHOP activations) and expression of other cellular stress molecules (JNK and c-Jun activations). Additionally, MLN4924 suppressed growth of CC xenografts in nude mice. Furthermore, we demonstrated that MLN4924 potentiated cisplatin-induced cytotoxicity in CC cells with activation of caspases. Consistently with this, MLN4924 significantly enhanced cisplatin-induced growth inhibition of CC xenografts. Together, these findings suggest that MLN4924 alone or in combination with cisplatin is of value in treating human CCs.

Patterns of target tissue reinnervation and trophic factor expression after nerve grafting.

BACKGROUND: Reinnervation of target tissues determines functional outcomes after nerve grafting, which is important in traumatic injury caused by accidents or consequences resulting from surgical removal of tumors. Previous studies documented the influences of nerve repair mainly based on nerve morphometry but rarely compared the final outcomes according to target reinnervation patterns by nerve fibers of different categories. METHODS: In a mouse model of nerve grafting, the authors analyzed the innervation indexes of different target tissues after transection-reimplantation on the sciatic nerve, which were defined as a parameter on the operated side normalized to that on the control side. RESULTS: Muscle reinnervation appeared to be the best compared with skin reinnervation (p < 0.0001) and sweat gland reinnervation (p < 0.0001) at postoperative month 3. The sudomotor reinnervation was relatively higher than the cutaneous reinnervation (p = 0.014). The abundance of trophin transcripts for brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin 3 (NT3) was higher in plantar muscles on the operated side than those on the control side. In contrast, transcripts of BDNF, GDNF, nerve growth factor, and NT3 were all similar in the footpad skin between the operated and control sides. CONCLUSIONS: The results suggested that, compared with the skin, muscles achieved the best reinnervation after nerve grafting, which was related to higher expression of BDNF, GDNF, and NT3 in muscles than in the skin.

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing.

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.

Sweet syndrome with histiocytoid infiltrate and neutropenia: a rare combination.

Histiocytoid Sweet syndrome is a recently described variant of acute febrile neutrophilic dermatosis. The histiocytoid cells are easily misinterpreted as histiocytes, when in fact they are immature myeloid cells. The recognition of myeloperoxidase expression in these cells is important in avoiding confusion with histiocyte-rich dermatoses. Herein, we report a case of histiocytoid Sweet syndrome with neutropenia that had an unusual presentation. The recognition of this rare combination helps expand the spectrum of Sweet syndrome with histiocytoid infiltrate.

DEN1 deneddylates non-cullin proteins in vivo.

The ubiquitin-like protein Nedd8/Rub1 covalently modifies and activates cullin ubiquitin ligases. However, the repertoire of Nedd8-modified proteins and the regulation of protein neddylation status are not clear. The cysteine protease DEN1/NEDP1 specifically processes the Nedd8 precursor and has been suggested to deconjugate Nedd8 from cullin proteins. By characterizing the Drosophila DEN1 protein and DEN1 null (DEN1(null)) mutants, we provide in vitro and in vivo evidence that DEN1, in addition to processing Nedd8, deneddylates many cellular proteins. Although purified DEN1 protein efficiently deneddylates the Nedd8-conjugated cullin proteins Cul1 and Cul3, neddylated Cul1 and Cul3 protein levels are not enhanced in DEN1(null). Strikingly, many cellular proteins are highly neddylated in DEN1 mutants and are deneddylated by purified DEN1 protein. DEN1 deneddylation activity is distinct from that of the cullin-deneddylating CSN. Genetic analyses indicate that a balance between neddylation and deneddylation maintained by DEN1 is crucial for animal viability.

Protection of cullin-RING E3 ligases by CSN-UBP12.

Neddylation, a process that conjugates the ubiquitin-like polypeptide NEDD8 to cullin proteins, activates cullin-RING ubiquitin ligases (CRLs). Deneddylation, in which the COP9 signalosome (CSN) removes NEDD8 from cullins, inactivates CRLs. However, genetic studies of CSN function conclude that deneddylation also promotes CRL activity. It has been proposed that a cyclic transition through neddylation and deneddylation is required for the regulation of CRL activity in vivo. Recent discoveries suggest that an additional level of complexity exists, whereby CRL components are targets for degradation, mediated either by autocatalytic ubiquitination or by unknown mechanisms. Deneddylation by CSN and deubiquitylation by CSN-associated ubiquitin-specific protease 12 protect CRL components from cellular depletion, thus maintaining the physiological CRL activities.

Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation.

Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN). Here we show that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. We further show that neddylated Cul1 and Cul3 are unstable - as suggested by the evidence that Nedd8 promotes the instability of both cullins - and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. We propose that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo.