NOTCH localizes to mitochondria through the TBC1D15-FIS1 interaction and is stabilized via blockade of E3 ligase and CDK8 recruitment to reprogram tumor-initiating cells.

The P53-destabilizing TBC1D15-NOTCH protein interaction promotes self-renewal of tumor-initiating stem-like cells (TICs); however, the mechanisms governing the regulation of this pathway have not been fully elucidated. Here, we show that TBC1D15 stabilizes NOTCH and c-JUN through blockade of E3 ligase and CDK8 recruitment to phosphodegron sequences. Chromatin immunoprecipitation (ChIP-seq) analysis was performed to determine whether TBC1D15-dependent NOTCH1 binding occurs in TICs or non-TICs. The TIC population was isolated to evaluate TBC1D15-dependent NOTCH1 stabilization mechanisms. The tumor incidence in hepatocyte-specific triple knockout (Alb::CreERT2;Tbc1d15Flox/Flox;Notch1Flox/Flox;Notch2Flox/Flox;HCV-NS5A) Transgenic (Tg) mice and wild-type mice was compared after being fed an alcohol-containing Western diet (WD) for 12 months. The NOTCH1-TBC1D15-FIS1 interaction resulted in recruitment of mitochondria to the perinuclear region. TBC1D15 bound to full-length NUMB and to NUMB isoform 5, which lacks three Ser phosphorylation sites, and relocalized NUMB5 to mitochondria. TBC1D15 binding to NOTCH1 blocked CDK8- and CDK19-mediated phosphorylation of the NOTCH1 PEST phosphodegron to block FBW7 recruitment to Thr-2512 of NOTCH1. ChIP-seq analysis revealed that TBC1D15 and NOTCH1 regulated the expression of genes involved in mitochondrial metabolism-related pathways required for the maintenance of TICs. TBC1D15 inhibited CDK8-mediated phosphorylation to stabilize NOTCH1 and protect it from degradation The NUMB-binding oncoprotein TBC1D15 rescued NOTCH1 from NUMB-mediated ubiquitin-dependent degradation and recruited NOTCH1 to the mitochondrial outer membrane for the generation and expansion of liver TICs. A NOTCH-TBC1D15 inhibitor was found to inhibit NOTCH-dependent pathways and exhibited potent therapeutic effects in PDX mouse models. This unique targeting of the NOTCH-TBC1D15 interaction not only normalized the perinuclear localization of mitochondria but also promoted potent cytotoxic effects against TICs to eradicate patient-derived xenografts through NOTCH-dependent pathways.

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells.

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs.

Tumor-initiating stem-like cells (TICs) are defective in maintaining asymmetric cell division and responsible for tumor recurrence. Cell-fate-determinant molecule NUMB-interacting protein (TBC1D15) is overexpressed and contributes to p53 degradation in TICs. Here we identify TBC1D15-mediated oncogenic mechanisms and tested the tumorigenic roles of TBC1D15 in vivo. We examined hepatocellular carcinoma (HCC) development in alcohol Western diet-fed hepatitis C virus NS5A Tg mice with hepatocyte-specific TBC1D15 deficiency or expression of non-phosphorylatable NUMB mutations. Liver-specific TBC1D15 deficiency or non-p-NUMB expression reduced TIC numbers and HCC development. TBC1D15-NuMA1 association impaired asymmetric division machinery by hijacking NuMA from LGN binding, thereby favoring TIC self-renewal. TBC1D15-NOTCH1 interaction activated and stabilized NOTCH1 which upregulated transcription of NANOG essential for TIC expansion. TBC1D15 activated three novel oncogenic pathways to promote self-renewal, p53 loss, and Nanog transcription in TICs. Thus, this central regulator could serve as a potential therapeutic target for treatment of HCC.

Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion.

Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2'-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase ß (polß) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polß incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.

Nanoparticle systems for cancer vaccine.

As effective tools for public health, vaccines prevent disease by priming the body's adaptive and innate immune responses against an infection. Due to advances in understanding cancers and their relationship with the immune system, there is a growing interest in priming host immune defenses for a targeted and complete antitumor response. Nanoparticle systems have shown to be promising tools for effective antigen delivery as vaccines and/or for potentiating immune response as adjuvants. Here, we highlight relevant physiological processes involved in vaccine delivery, review recent advances in the use of nanoparticle systems for vaccines and discuss pertinent challenges to viably translate nanoparticle-based vaccines and adjuvants for public use.

The Cancer Mutation D83V Induces an α-Helix to ß-Strand Conformation Switch in MEF2B.

MEF2B is a major target of somatic mutations in non-Hodgkin lymphoma. Most of these mutations are non-synonymous substitutions of surface residues in the MADS-box/MEF2 domain. Among them, D83V is the most frequent mutation found in tumor cells. The link between this hotspot mutation and cancer is not well understood. Here we show that the D83V mutation induces a dramatic α-helix to ß-strand switch in the MEF2 domain. Located in an α-helix region rich in ß-branched residues, the D83V mutation not only removes the extensive helix stabilization interactions but also introduces an additional ß-branched residue that further shifts the conformation equilibrium from α-helix to ß-strand. Cross-database analyses of cancer mutations and chameleon sequences revealed a number of well-known cancer targets harboring ß-strand favoring mutations in chameleon α-helices, suggesting a commonality of such conformational switch in certain cancers and a new factor to consider when stratifying the rapidly expanding cancer mutation data.

Synthesis of 14',15'-dehydro-ritterazine Y via reductive and oxidative functionalizations of hecogenin acetate.

An analog of ritterazine Y was synthesized from hecogenin acetate in 23 steps via functional group manipulations of hecogenin acetate. Preparation of the north G and south Y units and the late stage Guo-Fuchs asymmetric coupling of the both units afforded the ritterazine Y analog.

Involvement of nuclear factor kappa B in high-fat diet-related pancreatic fibrosis in rats.

BACKGROUND/AIMS: High-fat diets contribute to pancreatic fibrogenesis, but the pathogenesis remains unclear. This study investigated the role of nuclear factor kappa B (NF-κB) in high-fat diet-induced pancreatic fibrosis in rats. METHODS: Male Wistar rats were fed a high-fat diet or standard normal chow for 20 weeks. Pancreatic fibrosis was determined by Sirius red staining. Immunohistochemical staining, reverse transcription-polymerase chain reaction and Western blotting were used to identify NF-κB-associated genes or protein expressions. RESULTS: Inflammation, fat deposition, pancreatic stellate cell activation and fibrosis were observed in the pancreases of the high-fat diet group. NF-κB subunit p65 (NF-κB/p65) expression was localized to the nucleus, and intercellular adhesion molecule 1 (ICAM-1) was over-expressed. Pancreatic gene expression levels of NF-κB/p65, ICAM-1 and tumor necrosis factor α were all elevated significantly in rats fed a high-fat diet compared with control rats. Western blotting also revealed significantly increased levels of ICAM-1 and nuclear NF-κB/p65 in rats fed high-fat diets comparison with control rats. CONCLUSIONS: NF-κB is involved in high-fat diet-related pancreatic fibrosis.

Application of hypoiodite-mediated aminyl radical cyclization to synthesis of solasodine acetate.

Solasodine acetate, an anticancer steroidal alkaloid, was synthesized from diosgenin in 8 steps with an overall yield of 23%. A key synthetic step involves the formation of 5/6-oxazaspiroketal moiety via hypoiodite-mediated aminyl radical cyclization of a steroidal primary amine.

In trans interaction of hepatitis C virus helicase domains mediates protease activity critical for internal NS3 cleavage and cell transformation.

Hepatitis C virus (HCV) internal non-structural protein 3 (NS3) cleavage can occur in trans in the presence of NS4A. In this study, we have further demonstrated a critical role of the helicase domain in the internal NS3 cleavage, different from HCV polyprotein processing which requires only the serine protease domain. The NTPase domain of NS3 helicase interacts with the RNA binding domain to facilitate internal NS3 cleavage. In addition, NS3 protease activity contributes to the transforming ability of the major internal cleavage product NS3(1-402). These findings imply important roles of the internal cleavage and protease activity of the NS3 protein in the pathogenesis of HCV.

Differential requirements of NS4A for internal NS3 cleavage and polyprotein processing of hepatitis C virus.

The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT(402)|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.

Hepatitis C virus NS4A inhibits cap-dependent and the viral IRES-mediated translation through interacting with eukaryotic elongation factor 1A.

The genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS3 and NS2-3 proteases. Nonstructural protein 4A (NS4A) is a cofactor of the NS3 serine protease and has been demonstrated to inhibit protein synthesis. In this study, GST pull-down assay was performed to examine potential cellular factors that interact with the NS4A protein and are involved in the pathogenesis of HCV. A trypsin digestion followed by LC-MS/MS analysis revealed that one of the GST-NS4A-interacting proteins to be eukaryotic elongation factor 1A (eEF1A). Both the N-terminal domain of NS4A from amino acid residues 1-20, and the central domain from residues 21-34 interacted with eEF1A, but the central domain was the key player involved in the NS4A-mediated translation inhibition. NS4A(21-34) diminished both cap-dependent and HCV IRES-mediated translation in a dose-dependent manner. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. Taken together, NS4A inhibits host and viral translation through interacting with eEF1A, implying a possible mechanism by which NS4A is involved in the pathogenesis and chronic infection of HCV.

Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent.

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.

A phase IB clinical and pharmacokinetic study of the angiogenesis inhibitor SU5416 and paclitaxel in recurrent or metastatic carcinoma of the head and neck.

PURPOSE: SU5416 is a novel small organic molecule that non-competitively inhibits the phosphorylation of the VEGF tyrosine kinase receptor, Flk-1. This phase IB study was performed to determine the safety, pharmacokinetics, and preliminary efficacy of the combination of SU5416 and paclitaxel in recurrent or metastatic carcinoma of the head and neck. METHODS: Enrolled in the study were 12 patients with biopsy-proven recurrent or metastatic carcinoma of the head and neck. Six patients received intravenous SU5416 110 mg/m2 on days 1, 15, 18, 22 and 25, and paclitaxel 70 mg/m2 on days 8, 15 and 22. Since two patients experienced a dose-limiting toxicity (DLT) in cohort 1, the next six patients received identical treatment as above except the paclitaxel dose was reduced to 55 mg/m2 per week. RESULTS: A total of 42 cycles at two different dose levels were given. In cohort 1 there were two deep venous thromboses that were DLTs. In the second cohort there was a DLT consisting of a transient ischemic attack after receiving SU5416. Most of the other toxicities seen were grade 1 or 2 in nature and consisted of headache, facial flushing, and fatigue. Two patients developed extensive ulcerative cavities at sites of prior radiation. There were no significant changes in the pharmacokinetic parameters of SU5416 given with paclitaxel. Four patients had prolonged freedom from progression of 18, 28, 42, and 60 weeks duration. CONCLUSIONS: The combination of SU5416 with paclitaxel had a higher than expected incidence of thromboembolic events and prophylactic anticoagulation should be considered for future trials that combine an angiogenesis inhibitor with cytotoxic chemotherapy. Although the future development of SU5416 as a chemotherapeutic agent is unclear, there was a clinical benefit seen with this combination in 36% of the patients. This trial supports the use of developing antiangiogenic combinations, using molecular targeted agents, in head and neck carcinoma.

Ceramide metabolite, not intact ceramide molecule, may be responsible for cellular toxicity.

Ceramides, which are produced from the hydrolysis of sphingomyelin or synthesized from serine and palmitate in a de novo pathway, are regarded as important cellular signals for inducing apoptosis. However, controversy over this proposed role of ceramides exists. Using stable isotope labelling coupled with GC (gas chromatography)-MS and mass isotopomer distribution analysis, we have studied the metabolism of exogenous long-chain ceramides in HL60 cells. Our results do not support the concept of enhanced ceramide transport into cells induced by solvent mixtures of ethanol and hydrocarbons. In addition, cell toxicity does not correlate with the amount of intact ceramide in the cells. Our results are more consistent with a disturbance of sphingomyelin metabolism induced by the solvent mixture. The characteristics of this disturbed sphingolipid disposition are the inhibition of dihydroceramide desaturation and an enhanced degradation of sphingomyelin. As a consequence, dihydroceramides accumulate and the cellular sphingomyelin content decreases. Inhibition of these pathways is most likely to be induced by the increased production of novel ceramide metabolites instead of by intact ceramides. Octadecane-1,2-diol is identified as a possible mediator. Treatments that divert ceramide degradation to the novel pathway are potential strategies in cancer therapy for inducing cell toxicity.

Studies of lipid turnover in cells with stable isotope and gas chromatograph-mass spectrometry.

Phospholipids are major building blocks for biological membranes. In addition, metabolites derived from their degradation are important signals in major cellular events, such as proliferation and apoptosis. The concept of lipid signaling in cells is derived mainly from the measurement of change in the concentration of lipid molecules. However, these changes in concentration are only a small part of the underlying metabolic change induced by a perturbation in the cell. In contrast, metabolic kinetic studies documenting product-precursor relationships and turnover rates are useful in elucidating the responsible mechanisms. Historically, metabolic studies of phospholipids in cells have been carried out with pulse or pulse-chase methods using radioactive isotopes. While these studies provide valuable information, their scope is restricted by inherent limitations. In this paper we describe a method using [1,2,3,4-13C(4)]palmitate as the tracer for studying the metabolic kinetics of the molecular species of diacylglycerol, ceramide, phosphatidylcholine, and sphingomyelin. After growing cells in the presence of labeled palmitate complexed to serum albumin, the lipids are extracted and separated into lipid classes. After enzymatic hydrolysis, diacylglyerols and ceramides as bis-trimethylsilyl derivatives are determined quantitatively with capillary column gas chromatography. Internal standards for each lipid class are used in the procedure. In addition, the isotopic enrichments of the lipid molecular species are determined with gas chromatograph-mass spectrometry. We applied this method to the study of HL60 cells. Different turnover rates were found for various molecular species. In addition, the sn-1 and sn-2 acyl groups appear to be synthesized at different rates for different molecular species. Other information, such as chain elongation and desaturation, might also be derived through the use of this method.

Quantitation and molecular species determination of diacylglycerols, phosphatidylcholines, ceramides, and sphingomyelins with gas chromatography.

In addition to the role of building block for biological membranes, phospholipids and their metabolites have been implicated in other important cellular functions, such as proliferation and apoptosis. Ceramides and their precursor, sphingomyelin, are thought to play a role in cellular apoptosis. In contrast, the metabolism of diacylglycerols and one of their precursors, phosphatidylcholine, is thought to be partly responsible for the opposite effect, cellular proliferation. Quantitative determination of these lipids in biological samples is important in investigating the complicated interactions between these molecules. In this report, we describe a capillary gas chromatographic procedure for the quantitative determination of molecular species of diacylglycerols, ceramides, phosphatidylcholines, and sphingomyelins. Lipid extracts are separated into these classes with a silica gel column. Diacylglycerols and ceramides are analyzed as trimethylsilyl derivatives. Phosphatidylcholines and sphingomyelins are converted to their diacylglycerol and ceramide components with sphingomyelinase hydrolysis. Internal standards for each analyzed fraction are used in the procedure. This method is used to determine the lipids in liver homogenate and subcellular fractions, including mitochondria, light mitochondria, and microsomes from young and old Fischer 344 rats. Our data show that the ceramide and sphingomyelin content is higher in the mitochondria of old rats. This relationship is consistent with the potential role of ceramide in mitochondria-induced apoptosis. More study is needed to substantiate this relationship.

Pharmacokinetics of O6-benzylguanine (NSC637037) and its metabolite, 8-oxo-O6-benzylguanine.

O6-Benzylguanine and its metabolite, 8-oxo-O6-benzylguanine, are equally potent inhibitors of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase. Pharmacokinetic values are derived from cancer patients participating in a phase I trial (10 or 20 mg/m2 of O6-benzylguanine in a single bolus dose or 10 to 120 mg/m2 as a 60-min constant infusion). A two-compartment model fits the plasma concentration versus time profile of O6-benzylguanine. O6-Benzylguanine is eliminated rapidly from the plasma compartment in humans (t1/2 alpha and t1/2 beta are 2 +/- 2 min and 26 +/- 15 min [mean +/- SD, n = 7], respectively), and its plasma clearance (513 +/- 148 mL/min/m2) is not dose dependent. Metabolite kinetics are evaluated using both a novel approach describing the relationship between O6-benzylguanine and 8-oxo-O6-benzylguanine and classical metabolite kinetics methods. With increasing doses of O6-benzylguanine, the plasma clearance of 8-oxo-O6-benzylguanine, decreases, prolonging elimination of the metabolite. This effect is not altered by coadministration of BCNU. The urinary excretion of drug and metabolites is minimal.