MDA-9/syntenin: a positive gatekeeper of melanoma metastasis.

Melanoma differentiation associated gene-9 (MDA-9), synonymous with syntenin, is an adapter protein that provides a central role in regulating cell-cell and cell-matrix adhesion. MDA-9/syntenin transduces signals from the cell-surface to the interior through its interaction with a plethora of additional proteins and actively participates in intracellular trafficking and cell-surface targeting, synaptic transmission, and axonal outgrowth. Recent studies demarcate a seminal role of MDA-9/syntenin in cancer metastasis. In the context of melanoma, MDA-9/syntenin functions as a positive regulator of melanoma progression and metastasis through interactions with c-Src and promotes the formation of an active FAK/c-Src signaling complex leading to NF-k B and matrix metalloproteinase (MMP) activation. The present review provides a current perspective of our understanding of the important features of MDA-9/syntenin and its significant role in tumor cell metastasis with special focus on molecular mechanism of action.

Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics.

Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Analysis of the 2.5 kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many ß-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a ß-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes through the NF-κB signaling pathway. The NF-κB binding site at -272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.

Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells.

Inappropriate drug delivery, secondary toxicities, and persistent chemo- and immunoresistance have traditionally compromised treatment response in melanoma. Using cellular systems and genetically engineered mouse models, we show that melanoma cells retain an innate ability to recognize cytosolic double-stranded RNA (dsRNA) and mount persistent stress response programs able to block tumor growth, even in highly immunosuppressed backgrounds. The dsRNA mimic polyinosine-polycytidylic acid, coadministered with polyethyleneimine as carrier, was identified as an unanticipated inducer of autophagy downstream of an exacerbated endosomal maturation program. A concurrent activity of the dsRNA helicase MDA-5 driving the proapoptotic protein NOXA resulted in an efficient autodigestion of melanoma cells. These results reveal tractable links for therapeutic intervention among dsRNA helicases, endo/lysosomes, and apoptotic factors.

Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: key regulators of innate immunity.

The innate immune system responds within minutes of infection to produce type I interferons and pro-inflammatory cytokines. Interferons induce the synthesis of cell proteins with antiviral activity, and also shape the adaptive immune response by priming T cells. Despite the discovery of interferons over 50 years ago, only recently have we begun to understand how cells sense the presence of a virus infection. Two families of pattern recognition receptors have been shown to distinguish unique molecules present in pathogens, such as bacterial and fungal cell wall components, viral RNA and DNA, and lipoproteins. The first family includes the membrane-bound toll-like receptors (TLRs). Studies of the signaling pathways that lead from pattern recognition to cytokine induction have revealed extensive and overlapping cascades that involve protein-protein interactions and phosphorylation, and culminate in activation of transcription proteins that control the transcription of genes encoding interferons and other cytokines. A second family of pattern recognition receptors has recently been identified, which comprises the cytoplasmic sensors of viral nucleic acids, including MDA-5, RIG-I, and LGP2. In this review we summarize the discovery of these cytoplasmic sensors, how they recognize nucleic acids, the signaling pathways leading to cytokine synthesis, and viral countermeasures that have evolved to antagonize the functions of these proteins. We also consider the function of these cytoplasmic sensors in apoptosis, development and differentiation, and diabetes.

RIG-I is cleaved during picornavirus infection.

The innate immune system senses RNA virus infections through membrane-bound Toll-like receptors or the cytoplasmic proteins RIG-I and MDA-5. RIG-I is believed to recognize the 5'-triphosphate present on many viral RNAs, and hence is important for sensing infections by paramyxoviruses, influenza viruses, rhabdoviruses, and flaviviruses. MDA-5 recognizes dsRNA, and senses infection with picornaviruses, whose RNA 5'-ends are linked to a viral protein, VPg, not a 5'-triphosphate. We previously showed that MDA-5 is degraded in cells infected with different picornaviruses, and suggested that such cleavage might be a mechanism to antagonize production of type I IFN in response to viral infection. Here we examined the state of RIG-I during picornavirus infection. RIG-I is degraded in cells infected with poliovirus, rhinoviruses, echovirus, and encephalomyocarditis virus. In contrast to MDA-5, cleavage of RIG-I is not accomplished by cellular caspases or the proteasome. Rather, the viral proteinase 3C(pro) cleaves RIG-I, both in vitro and in cells. Cleavage of RIG-I during picornavirus infection may constitute another mechanism for attenuating the innate response to viral infection.

Phagocytosis of picornavirus-infected cells induces an RNA-dependent antiviral state in human dendritic cells.

Dendritic cells (DCs) play a central role in instructing antiviral immune responses. DCs, however, can become targeted by different viruses themselves. We recently demonstrated that human DCs can be productively infected with echoviruses (EVs), but not coxsackie B viruses (CVBs), both of which are RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. We now show that phagocytosis of CVB-infected, type I interferon-deficient cells induces an antiviral state in human DCs. Uptake of infected cells increased the expression of the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5 as well as other interferon-stimulated genes and protected DCs against subsequent infection with EV9. These effects depended on recognition of viral RNA and could be mimicked by exposure to the synthetic double-stranded RNA analogue poly(I:C) but not other Toll-like receptor (TLR) ligands. Blocking endosomal acidification abrogated protection, suggesting a role for TLRs in the acquisition of an antiviral state in DCs. In conclusion, recognition of viral RNA rapidly induces an antiviral state in human DCs. This might provide a mechanism by which DCs protect themselves against viruses when attracted to an environment with ongoing infection.

mda-7/IL-24, novel anticancer cytokine: focus on bystander antitumor, radiosensitization and antiangiogenic properties and overview of the phase I clinical experience (Review).

Subtraction hybridization applied to a 'differentiation therapy' model of cancer employing human melanoma cells resulted in the cloning of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). Initial studies confirm an inverse correlation between mda-7 expression and melanoma development and progression. Forced expression of mda-7 by means of a plasmid or via a replication incompetent adenovirus (Ad.mda-7) promotes growth suppression and induces apoptosis in a broad array of human cancers. In contrast, mda-7 does not induce growth suppressive or toxic effects in normal cells. Based on structure (containing an IL-10 signature motif), secretion by cells (including subsets of T-cells) and location on chromosome 1q (in an area containing IL-10-family genes), mda-7 has now been renamed mda-7/IL-24. Studies by several laboratories have uncovered many of mda-7/IL-24's unique properties, including cancer-specific apoptosis-induction, cell cycle regulation, an ability to inhibit angiogenesis, potent 'bystander antitumor activity' and a capacity to enhance the sensitivity of tumor cells to radiation, chemotherapy and monoclonal antibody therapy. Moreover, based on its profound cancer tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant clinical activity. In these contexts, mda-7/IL-24 represents a unique cytokine gene with potential for therapy of human cancers. The present review focuses on three unique properties of mda-7/IL-24, namely its potent 'bystander antitumor activity', ability to sensitize tumor cells to radiation, and its antiangiogenesis properties. Additionally, an overview of the phase I clinical trial is provided. These studies affirm that mda-7/IL-24 has promise for the management of diverse cancers.

Central role of interferon regulatory factor-1 (IRF-1) in controlling retinoic acid inducible gene-I (RIG-I) expression.

Retinoic acid inducible gene-I (RIG-I) functions as the first line of defense against viral infection by sensing dsRNA and inducing type I interferon (IFN) production. The expression of RIG-I itself is induced by IFN-alpha/beta and dsRNA. To comprehend the molecular mechanism of expression regulation, we cloned the RIG-I promoter and analyzed its activity upon IFN-beta and dsRNA treatment. Under basal condition, RIG-I mRNA level and promoter activity were significantly higher in normal cells versus their tumor counterparts. In both normal and cancer cells, RIG-I expression was induced by IFN-beta and dsRNA. A single IRF-1 binding site in the proximal promoter functioned as a crucial regulator of basal, IFN-beta- and dsRNA-mediated induction of the RIG-I promoter. IFN-beta and dsRNA treatment increased IRF-1 binding to the RIG-I promoter. IRF-1 expression was also higher in normal cells than in cancer cells and it was induced by IFN-beta with similar kinetics as RIG-I. These results confirm that by controlling RIG-I expression, IRF-1 plays an essential role in anti-viral immunity. IRF-1 is a tumor suppressor and the expression profile of RIG-I together with its regulation by IRF-1 and the presence of a caspase-recruitment domain in RIG-I suggest that RIG-I might also possess tumor suppressor properties.

The effect of CtBP1 binding on the structure of the C-terminal region of adenovirus 12 early region 1A.

Adenovirus early region 1A (AdE1A) binds to the C-terminal binding protein 1 (CtBP1) primarily through a highly conserved PXDLS motif located close to its C-terminus. Purified synthetic peptides equivalent to this region of AdE1A have been shown to form a series of beta-turns. In this present study the effect of CtBP1 binding on the conformation of C-terminal region of Ad12E1A has been investigated. Using one- and two-dimensional (1)H NMR spectroscopy, the conformation of 20-residue peptides equivalent to amino acids I(241)-V(260) and E(247)-N(266) of Ad12E1A were examined in the absence of CtBP1. Whilst the latter peptide forms a series of beta-turns in its C-terminal half as reported previously, the former peptide is alpha-helical over the region D(243)-Q(253). Upon interaction with CtBP1 the conformation of the backbone in the region (255)PVDLCVK(261) of the Ad12E1A E(247)-N(266) peptide reorganises from a predominately beta-turn to an alpha-helical conformation. This structural isomerisation is characterised by a shift upfield of 0.318 ppm for the delta-CH(3) proton resonance of V(256). 2-D NOESY experiments showed new signals in the amide-alpha region which correlate to transferred NOEs from the protein to the peptide residues E(251), V(256) and K(261). In further analyses the contribution of individual amino acids within the sequence (254)VPVDLS(259) was assessed for their importance in determining structure and consequently affinity of the peptide for CtBP. It has been concluded that Ad12E1A residues (255)P-V(260) serve initially as a recognition site for CtBP and then as an anchor through a beta-turns-->alpha-helix conformational rearrangement. In addition it has been predicted that regions N-terminal to the PXDLS motif in AdE1As from different virus serotypes and from mammalian proteins form alpha-helices.

MDA-5 is cleaved in poliovirus-infected cells.

Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4 h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16 or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2Apro or 3Cpro. Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis. Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells treated with puromycin is approximately 90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells. Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral infection.

The interaction of the hnRNP family member E1B-AP5 with p53.

Adenovirus early region 1B-associated protein 5, E1B-AP5, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, was originally isolated on the basis of its ability to bind to the adenovirus 5 early region1B55K protein. Here, it has been demonstrated that E1B-AP5 interacts with mutant and wild-type p53 from human cells in pull-down assays using GST-E1B-AP5. This interaction has been confirmed by co-immunoprecipitation studies and pull-down experiments with in vitro translated E1B-AP5 and GST-p53. The binding site for E1B-AP5 has been mapped to the C-terminal region of p53. In reciprocal experiments, it has been shown that several regions of E1B-AP5 bound to p53 although it is probable that a major site of interaction is located between amino acids 395 and 732 of E1B-AP5. In reporter assays, E1B-AP5 inhibited p53 transcriptional activity although not as efficiently as the Ad5E1B55K protein. Transfection of E1B-AP5 into human tumour cells affected the cellular response to UV radiation, such that, although p53 expression was induced, little change in the level of p53-inducible genes could be observed.