Mutant Huntingtin impairs post-Golgi trafficking to lysosomes by delocalizing optineurin/Rab8 complex from the Golgi apparatus

Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.

Calcineurin inhibitors activate the proto-oncogene Ras and promote protumorigenic signals in renal cancer cells.

The development of cancer is a major problem in immunosuppressed patients, particularly after solid organ transplantation. We have recently shown that calcineurin inhibitors (CNI) used to treat transplant patients may play a critical role in the rapid progression of renal cancer. To examine the intracellular signaling events for CNI-mediated direct tumorigenic pathway(s), we studied the effect of CNI on the activation of proto-oncogenic Ras in human normal renal epithelial cells (REC) and renal cancer cells (786-0 and Caki-1). We found that CNI treatment significantly increased the level of activated GTP-bound form of Ras in these cells. In addition, CNI induced the association of Ras with one of its effector molecules, Raf, but not with Rho and phosphatidylinositol 3-kinase; CNI treatment also promoted the phosphorylation of the Raf kinase inhibitory protein and the downregulation of carabin, all of which may lead to the activation of the Ras-Raf pathway. Blockade of this pathway through either pharmacologic inhibitors or gene-specific small interfering RNA significantly inhibited CNI-mediated augmented proliferation of renal cancer cells. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the Ras-Raf pathway is significantly activated in the tumor tissues of CNI-treated mice. Together, targeting the Ras-Raf pathway may prevent the development/progression of renal cancer in CNI-treated patients.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1alpha (HIF-1alpha) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1alpha and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1alpha inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1alpha resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phospho-STAT3 and HIF-1alpha are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1alpha in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

mTORC2 regulates PGE(2)-mediated endothelial cell survival and migration

Prostaglandin E-2 (PGE(2)) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE(2)-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE(2)-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE2-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE2-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE2-mediated endothelial cell responses.

CDK9 regulates AR promoter selectivity and cell growth through serine 81 phosphorylation.

Previously we determined that S81 is the highest stoichiometric phosphorylation on the androgen receptor (AR) in response to hormone. To explore the role of this phosphorylation on growth, we stably expressed wild-type and S81A mutant AR in LHS and LAPC4 cells. The cells with increased wild-type AR expression grow faster compared with parental cells and S81A mutant-expressing cells, indicating that loss of S81 phosphorylation limits cell growth. To explore how S81 regulates cell growth, we tested whether S81 phosphorylation regulates AR transcriptional activity. LHS cells stably expressing wild-type and S81A mutant AR showed differences in the regulation of endogenous AR target genes, suggesting that S81 phosphorylation regulates promoter selectivity. We next sought to identify the S81 kinase using ion trap mass spectrometry to analyze AR-associated proteins in immunoprecipitates from cells. We observed cyclin-dependent kinase (CDK)9 association with the AR. CDK9 phosphorylates the AR on S81 in vitro. Phosphorylation is specific to S81 because CDK9 did not phosphorylate the AR on other serine phosphorylation sites. Overexpression of CDK9 with its cognate cyclin, Cyclin T, increased S81 phosphorylation levels in cells. Small interfering RNA knockdown of CDK9 protein levels decreased hormone-induced S81 phosphorylation. Additionally, treatment of LNCaP cells with the CDK9 inhibitors, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole and Flavopiridol, reduced S81 phosphorylation further, suggesting that CDK9 regulates S81 phosphorylation. Pharmacological inhibition of CDK9 also resulted in decreased AR transcription in LNCaP cells. Collectively these results suggest that CDK9 phosphorylation of AR S81 is an important step in regulating AR transcriptional activity and prostate cancer cell growth.

Herbivory in the previous generation primes plants for enhanced insect resistance.

Inducible defenses, which provide enhanced resistance after initial attack, are nearly universal in plants. This defense signaling cascade is mediated by the synthesis, movement, and perception of jasmonic acid and related plant metabolites. To characterize the long-term persistence of plant immunity, we challenged Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) with caterpillar herbivory, application of methyl jasmonate, or mechanical damage during vegetative growth and assessed plant resistance in subsequent generations. Here, we show that induced resistance was associated with transgenerational priming of jasmonic acid-dependent defense responses in both species, caused caterpillars to grow up to 50% smaller than on control plants, and persisted for two generations in Arabidopsis. Arabidopsis mutants that are deficient in jasmonate perception (coronatine insensitive1) or in the biogenesis of small interfering RNA (dicer-like2 dicer-like3 dicer-like4 and nuclear RNA polymerase d2a nuclear RNA polymerase d2b) do not exhibit inherited resistance. The observation of inherited resistance in both the Brassicaceae and Solanaceae suggests that this trait may be more widely distributed in plants. Epigenetic resistance to herbivory thus represents a phenotypically plastic mechanism for enhanced defense across generations.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Adiponectin and heme oxygenase-1 suppress TLR4/MyD88-independent signaling in rat Kupffer cells and in mice after chronic ethanol exposure.

Alcoholic liver disease is mediated via activation of TLR4 signaling; MyD88-dependent and -independent signals are important contributors to injury in mouse models. Adiponectin, an anti-inflammatory adipokine, suppresses TLR4/MyD88-dependent responses via induction of heme oxygenase-1 (HO-1). Here we investigated the interactions between chronic ethanol, adiponectin, and HO-1 in regulation of TLR4/MyD88-independent signaling in macrophages and an in vivo mouse model. After chronic ethanol feeding, LPS-stimulated expression of IFN-β and CXCL10 mRNA was increased in primary cultures of Kupffer cells compared with pair-fed control mice. Treatment of Kupffer cells with globular adiponectin (gAcrp) normalized this response. LPS-stimulated IFN-β/CXCL10 mRNA and CXCL10 protein was also reduced in RAW 264.7 macrophages treated with gAcrp or full-length adiponectin. gAcrp and full-length adiponectin acted via adiponectin receptors 1 and 2, respectively. gAcrp decreased TLR4 expression in both Kupffer cells and RAW 264.7 macrophages. Small interfering RNA knockdown of HO-1 or inhibition of HO-1 activity with zinc protoporphyrin blocked these effects of gAcrp. C57BL/6 mice were exposed to chronic ethanol feeding, with or without treatment with cobalt protoporphyrin, to induce HO-1. After chronic ethanol feeding, mice were sensitized to in vivo challenge with LPS, expressing increased IFN-β/CXCL10 mRNA and CXCL10 protein in liver compared with control mice. Pretreatment with cobalt protoporphyrin 24 h before LPS challenge normalized this effect of ethanol. Adiponectin and induction of HO-1 potently suppressed TLR4-dependent/MyD88-independent cytokine expression in primary Kupffer cells from rats and in mouse liver after chronic ethanol exposure. These data suggest that induction of HO-1 may be a useful therapeutic strategy in alcoholic liver disease.

Ophtalmologie. Thérapies anti-vasoprolifératives

The most important recent advance in the treatment of neovascular age-related macular degeneration (AMD) is the development of antivascular endothelial growth factor (anti-VEGF) therapeutic agents that preserve and improve visual acuity by arresting choroidal neovascular growth and reducing vascular permeability. Two anti-VEGF agents, ranibizumab and pegaptanib sodium, are currently approved by Swissmedic for the treatment of neovascular AMD. A third anti-VEGF agent, bevacizumab, is currently used as an off label treatment option for exsudative AMD. Other anti-VEGF agent strategies that have shown efficacy include among others, small interfering RNA agents to silence the VEGF gene and receptor and the fusion protein VEGF trap. Anti-VEGF therapies have been used successfully in the clinic, encouraging their use in the treatment of other neovascular and exudative eye diseases.

Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.

Comprehensive Genomic Characterization of Long Non-coding RNAs across Human Cancers.

The discovery of long non-coding RNA (lncRNA) has dramatically altered our understanding of cancer. Here, we describe a comprehensive analysis of lncRNA alterations at transcriptional, genomic, and epigenetic levels in 5,037 human tumor specimens across 13 cancer types from The Cancer Genome Atlas. Our results suggest that the expression and dysregulation of lncRNAs are highly cancer type specific compared with protein-coding genes. Using the integrative data generated by this analysis, we present a clinically guided small interfering RNA screening strategy and a co-expression analysis approach to identify cancer driver lncRNAs and predict their functions. This provides a resource for investigating lncRNAs in cancer and lays the groundwork for the development of new diagnostics and treatments.

Inhibition of cytohesin-1 by siRNA leads to reduced IGFR signaling in prostate cancer

To explore how cytohesin-1 (CYTH-1) small interfering RNA (siRNA) influences the insulin-like growth factor receptor (IGFR)-associated signal transduction in prostate cancer, we transfected human prostate cancer PC-3 cell lines with liposome-encapsulatedCYTH-1 siRNA in serum-free medium and exposed the cells to 100 nM IGF-1. The mRNA and protein levels of the signal molecules involved in the IGFR signaling pathways were determined by real-time PCR and detected by Western blotting. The relative mRNA levels of CYTH-1, c-Myc, cyclinD1 and IGF-1R (CYTH-1 siRNA group vs scrambled siRNA group) were 0.26 vs 0.97, 0.34 vs 1.06, 0.10 vs 0.95, and 0.27 vs 0.41 (P < 0.05 for all), respectively. The relative protein levels of CYTH-1, pIGF-1R, pIRS1, pAkt1, pErk1, c-Myc, and cyclinD1 (CYTH-1 siRNA group vsscrambled siRNA group) were 0.10 vs 1.00 (30 min), 0.10 vs 0.98 (30 min), 0.04 vs 0.50 (30 min), 0.10 vs 1.00 (30 min), 0.10 vs 1.00 (30 min), 0.13 vs 0.85 (5 h), and 0.08 vs 0.80 (7 h), respectively. The tyrosine kinase activity of IGF-1R was associated with CYTH-1. The proliferative activity of PC-3 cells transfected with CYTH-1 siRNA was significantly lower than that of cells transfected with scrambled siRNA at 48 h (40.5 vs87.6%, P < 0.05) and at 72 h (34.5 vs 93.5%, P < 0.05). In conclusion, the interference of siRNA with cytohesin-1 leads to reduced IGFR signaling in prostate cancer; therefore, CYTH-1 might serve as a new molecular target for the treatment of prostate cancer.

REGγ is a strong candidate for the regulation of cell cycle, proliferation and the invasion by poorly differentiated thyroid carcinoma cells

REGγ is a proteasome activator that facilitates the degradation of small peptides. Abnormally high expression of REGγ has been observed in thyroid carcinomas. The purpose of the present study was to explore the role of REGγ in poorly differentiated thyroid carcinoma (PDTC). For this purpose, small interfering RNA (siRNA) was introduced to down-regulate the level of REGγ in the PDTC cell line SW579. Down-regulation of REGγ at the mRNA and protein levels was confirmed by RT-PCR and Western blot analyses. FACS analysis revealed cell cycle arrest at the G1/S transition, the MTT assay showed inhibition of cell proliferation, and the Transwell assay showed restricted cell invasion. Furthermore, the expression of the p21 protein was increased, the expression of proliferating cell nuclear antigen (PCNA) protein decreased, and the expression of the p27 protein was unchanged as shown by Western blot analyses. REGγ plays a critical role in the cell cycle, proliferation and invasion of SW579 cells. The alteration of p21 and PCNA proteins related to the down-regulation of REGγ suggests that p21 and PCNA participate in the process of REGγ regulation of cell cycle progression and cell proliferation. Thus, targeting REGγ has a therapeutic potential in the management of PDTC patients.

Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro

Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells

Neuroblastoma is a solid tumor that occurs mainly in children. Malignant neuroblastomas have a poor prognosis because conventional chemotherapeutic agents are not very effective. Survivin, a member of the inhibitor of the apoptosis protein family, plays a significant role in cell division, inhibition of apoptosis, and promotion of cell proliferation and invasion. Previous studies found that survivin is highly expressed in some malignant neuroblastomas and is correlated with poor prognosis. The aim of this study was to investigate whether survivin could serve as a potential therapeutic target of human neuroblastoma. We employed RNA interference to reduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzed the effect of RNA interference on cell proliferation and invasion in vitro and in vivo. RNA interference of survivin led to a significant decrease in invasiveness and proliferation and increased apoptosis in SH-SY5Y cells in vitro. RNA interference of survivin inhibited tumor growth in vivo by 68±13% (P=0.002) and increased the number of apoptotic cells by 9.8±1.2% (P=0.001) compared with negative small interfering RNA (siRNA) treatment controls. Moreover, RNA interference of survivin inhibited the formation of lung metastases by 92% (P=0.002) and reduced microvascular density by 60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivin mRNA and protein expression both in vitro and in vivo compared with negative siRNA treatment controls. RNA interference of survivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasis formation. These results support further clinical development of RNA interference of survivin as a treatment of neuroblastoma and other cancer types.